Dissociated Tumor Cells Research Articles

Abstract 1375: Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy

Abstract Background: Intra-tumoral CD4+ and CD8+ T cells expressing inhibitory receptor CD161 have an effector memory phenotype with high cytotoxic potential. CD161 inhibits T and NK cell function in the presence of its ligand, CLEC2D. This suppression can be recovered by treatment with IMT-009, a first in class anti-CD161 antibody. Single cell transcriptomic analysis of anti-PD1 refractory microsatellite stable colorectal tumors identified presence of CD161+ CD4+ cytotoxic T cells and CD161+ PD1+ exhausted CD8 T cells suggesting that anti-CD161 treatment may provide benefit in these tumors both as a monotherapy and in combination with anti-PD1. It was also observed that CD161+ CD4/CD8T cells are more abundant in anti-PD1/L1 non responders compared with responders in hepatocellular carcinoma, cutaneous squamous cell carcinoma, clear cell renal carcinoma and breast cancer, providing a rationale for targeting CD161. Using engineered ex-vivo tumor models and dissociated human tumors, we have demonstrated that IMT-009 reverses CLEC2D mediated inhibition and restores primary T cell function. Additionally, co-treatment of IMT-009 with anti-PD1 further enhanced T cell mediated cytotoxicity, providing rationale for studies combining IMT-009 and anti PD1 in the clinic. Methods: Using the Snakemake pipeline developed in house, we processed publicly available scRNAseq data. Cells were clustered by Louvian method followed by cell type annotation by Seurat, and gene expression and gene signature levels per cluster were plotted. For ex-vivo assays, antigen specific CD8 T cells expanded from HLA-A2:01 donors were co-cultured with HLA-A2:01 expressing MDA-MB 231 triple negative breast cancer cell line or Raji Burkitt Lymphoma model with HLA-A2:01 allelic overexpression. Target tumor cells were engineered to express the ligands CLEC2D or PDL1. These cell lines were made to overexpress luciferase for monitoring target cell death using a luminescence readout. Additionally, dissociated tumor cells (DTCs) were cultured in presence of the treatment molecules. Intracellular staining of cytotoxic T cells along with detection of IFNγ, IL-2, TNFα and Granzyme B levels in culture supernatants by MSD/ELISA were used to assess activation profile of tumor infiltrated T cells. Results: Combining anti-PD1 and IMT-009 significantly enhanced cytotoxic benefit compared to individual monotherapy in both Raji and MDA-MB-231 target cell line models. IFNγ levels in supernatants of DTCs treated with this combination were elevated as compared to individual monotherapy. Together with scRNAseq data of increased abundance of KLRB1 (CD161) expressing T cells in anti PD1 non-responder patients, our data provide rationale for testing combination of IMT-009 with anti-PD1 in patients that may not respond to anti PD1 alone. Citation Format: Adwitiya Kar, Tim Nieuwenhuis, Meixue Duan, Siamak Tabrizi, Matthew Huggins, Elizabeth Scanlon, Cynthia Hession, Matthew Bernstein, Michael Ross, Ming Tang, Shruti Malu. Abundance of KLRB1+ (CD161) T cells in anti-PD1 non responders coupled with enhanced tumor cytotoxicity of anti-CD161 (IMT-009) with anti-PD1 makes it a rational target for combination with anti-PD-(L)1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1375.

Abstract 2500: Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine

Abstract Introduction: Next generation sequencing (NGS) has revolutionized precision oncology by enabling identification of cancer patients harboring actionable mutations who may benefit from corresponding targeted therapies. However, sequencing-based biomarkers are not detected for most patients and imperfectly predict response to therapy even when present. To address this unmet need, we have developed a CRISPR-based diagnostics platform for ‘personalized functional genomics’, allowing identification of patient-specific therapeutic vulnerabilities irrespective of mutation status. We have previously applied this approach to primary hematologic malignancies, and here report application to solid tumor-derived samples. Methods: Patient-derived solid tumor samples from several tumor types including breast, ovarian, colon and lung were obtained from 3 sources: fresh tissue (obtained from patients via core biopsy, surgical resection, or malignant fluid sampling), primary Patient-Derived Cell Lines (PDCLs, generated from surgical material and adapted for ex vivo culture), and commercially obtained viably frozen dissociated tumor cells (DTCs). Samples were dissociated to single cell suspensions and transduced with a lentivirus encoding Cas9 and an sgRNA library covering a custom targeted oncology gene panel. The panel consists of genes encoding targets of FDA-approved oncology drugs, as well as reference genes. After a short culture period, transduced cells were harvested and relative sgRNA distribution was measured by amplicon NGS. Changes in sgRNA abundance were collapsed into gene-level log2-fold changes, and significantly depleted genes scored as functional dependencies. For select PDCLs, cells were treated with cognate small molecule inhibitors over the course of 4 days. Cell viability was read out using CTG assay and a calculated IC50 lower than 500nM inferred as inhibitor sensitive. Results: In a double-blind study using primary PDCLs, functional dependencies identified by CRISPR perfectly discriminated alpelisib sensitive (2/2) from resistant samples (6/6). Using fresh primary samples from varying cancer types and specimen sources, we achieve a technical success rate >70%. Among gene dependencies identified, targets of FDA-approved oncology drugs and those under late-stage clinical investigation were both observed including BRAF, CDK4/6, VEGFR1/2 and PARP1/2. Conclusion: We demonstrate technical proof of concept of the FX platform for CRISPR-based personalized functional genomics. This approach is feasible when applied to primary patient samples from a variety of tumor types and tissue sources, and results are validated by comparison with orthogonal methods. CRISPR-based functional profiling represents a powerful adjunct to traditional genomic profiling, for the first time allowing drug target dependency mapping in a patient-specific and mutation-agnostic manner. Citation Format: Karl Hodel, Andreas Varkaris, Nicholas Chevalier, Maci Meyers, Allison Kehlmann, Stephane Corbel, Anastasia Stavridi, Peter Kilfeather, Leonardo Hagmann, Kathleen Tran, Yali Li, Christian Schmedt, Ioannis Sanidas, Srinath Sampath, Srihari Sampath, Dejan Juric. Technical feasibility of CRISPR-based functional profiling for solid tumor precision medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2500.

Abstract 6892: Murine ex vivo platform: A powerful tool for biomarker assessment and high throughput screening of translationally relevant in vivo tumor models for PD and efficacy studies

Abstract Ex vivo model systems using tumor biopsies from patients are often utilized to develop predictive biomarkers. However, these readouts are often proximal and do not inform on downstream mechanisms (distal pharmacodynamics) and their relationship to anti-tumor efficacy. In vivo murine models represent a powerful tool to bridge this gap. However, selection of an appropriate translationally relevant murine model can often be a time-consuming process given that the complexity of the tumor microenvironment (TME). Additionally, in vivo models are generally not amenable to high throughput screening. Here, we demonstrate that ex vivo mouse tumor platforms can be used for rapid screening of treatment responsive and translationally relevant tumor models. These models can be used to interrogate the impact of a therapeutic on anti-tumor efficacy in a long-term in vivo study, thus allowing for the establishment of biomarker/response relationships in a rational and rapid manner. To operationalize this platform, ex vivo cultures of dissociated tumor cells (DTC) or tumor tissue slices (histoculture) were first optimized for maximum viability and/or preservation of tissue architecture. To allow for high throughput screening, feasibility of freezing DTCs or tumor chunks prior to sectioning was also explored. Finally, both ex vivo tumor cultures and in vivo tumors were treated with a Innate agonist as a proof of concept to demonstrate functional equivalency. In conclusion, this work provides a novel way of screening responsive, translationally relevant murine tumor models in a high throughput and cost-effective manner. These efforts have the potential benefits of saving time and animal usage, while also maximizing the probability of successfully evaluating a therapeutic’s benefit. Citation Format: Sunitha Bachawal, Janet Leung, Remie Mandawe, Malia Garza, Kimberly ly, Ofir Stefanson, Jack Lohre, Diba Kamali, Ngan Doan, Benjamin King, Elliot Kim, Ganapathi Hegde, Xiaoyun Liao, Scot Liu, Jeong Kim, Amanda Mikels Vigdal, Anandaroop Mukhopadhyay. Murine ex vivo platform: A powerful tool for biomarker assessment and high throughput screening of translationally relevant in vivo tumor models for PD and efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6892.

Abstract 4072: Unleashing natural IL-18 activity using an anti-IL-18BP blocker antibody induces potent immune stimulation and anti-tumor effects

Abstract IL-18 is an inflammasome induced proinflammatory cytokine that augments T and NK cell activity and stimulates IFNγ production. The activity of IL-18 is naturally blocked by a high affinity endogenous binding protein (IL-18BP). IL-18BP is induced in the tumor microenvironment (TME) in response to IFNγ upregulation in a negative feedback mechanism. By evaluating 88 human tumor specimens and serum samples we were able to show that IL-18 is upregulated in the TME (median 11.2ng/gr) compared to serum samples (median 0.3ng/ml). Moreover, we showed that most of the IL-18 is bound by IL-18BP. IL-18BP-bound IL-18 levels were largely above the amount required for T cell activation in vitro (1.2ng/gr), implying that blocking IL-18BP has the potential to release IL-18 in tumors above the minimum range required for immune system stimulation. Next, to assess whether tumor endogenous IL-18 levels released by IL-18BP blockade are sufficient to provoke anti-tumor responses, COM503, a high affinity (<1pM) anti-IL-18BP Ab, was generated and examined in T and NK cell-based assays. In a co-culture assay of tumor cells with ex-vivo stimulated human tumor infiltrating CD8+ lymphocytes, COM503 was able to displace IL-18 from a pre-formed complex and enhance IFNγ (197%, p<0.01) and TNFα (84% p<0.01) secretion. Additionally, COM503 induced human NK cell activation as reflected by increased IFNγ secretion (26-fold, p<0.001). Finally, in human ex vivo dissociated tumor cells assay, COM503 mediated an increase in Granzyme B (25%), IFNγ (38%), TNFα (58%) and IL-12 (50%) production. In vivo, administration of an anti-mouse IL-18BP Ab resulted in potent anti-tumor responses and increased survival across multiple mouse tumor models. In orthotopic E0771 tumor model, anti-IL-18BP Ab induced significant tumor growth inhibition (91% TGI, p<0.0001), as well as pronounced TME-localized immune modulation. This modulation included an increase in CD8+ T cells expansion (108.5%, p=0.015) and activation, specifically in polyfunctional effector IFNγ+GrB+CD8+ T cells (259%, p=0.02) and IFNγ+TNFα+NK cells (77%, p=0.001). Similarly, anti-tumor effects were shown in MC38OVAdim model (58% TGI, p<0.001), accompanied by a robust TME-localized immune modulation including increased CD8+ T cells expansion (85%, p=0.009) and IFNγ secretion (76%, p=0.052). In contrast to immune modulation in the TME, no increase in inflammatory cytokines and lymphocyte numbers or activation state was observed in serum and spleen. Taken together, our data suggest that IL-18 is upregulated in the TME, mostly bound by IL-18BP, and could be exploited to induce pronounced TME-localized immune modulation. Anti-IL-18BP Ab approach has a leading edge in inhibiting tumor growth while avoiding peripheral toxicity associated with administration of a cytokine. COM503 is currently undergoing IND-enabling studies. Citation Format: Assaf Menachem, Zoya Alteber, Gady Cojocaru, Tal Fridman Kfir, Dan Blat, Olga Leiderman, Moran Galperin, Lital Sever, Nadav Cohen, Keren Cohen, Roy Zvi Granit, Sandra Vols, Masha Frenkel, Lior Faigenbloom, Liron Soffer, Karin Meyer, Keren Menachem, Hadas Galon Tilleman, Dina Morein, Itamar Borukhov, Amir Toporik, Michal Perpinial Shahor, Evgeny Tatirovsky, Pierre Ferre, Eran Ophir. Unleashing natural IL-18 activity using an anti-IL-18BP blocker antibody induces potent immune stimulation and anti-tumor effects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4072.

Toripalimab, a therapeutic monoclonal anti-PD-1 antibody with high binding affinity to PD-1 and enhanced potency to activate human T cells

Over the past decade, US Food and Drug Administration (FDA)-approved immune checkpoint inhibitors that target programmed death-1 (PD-1) have demonstrated significant clinical benefit particularly in patients with PD-L1 expressing tumors. Toripalimab is a humanized anti-PD-1 antibody, approved by FDA for first-line treatment of nasopharyngeal carcinoma in combination with chemotherapy. In a post hoc analysis of phase 3 studies, toripalimab in combination with chemotherapy improved overall survival irrespective of PD-L1 status in nasopharyngeal carcinoma (JUPITER-02), advanced non-small cell lung cancer (CHOICE-01) and advanced esophageal squamous cell carcinoma (JUPITER-06). On further characterization, we determined that toripalimab is molecularly and functionally differentiated from pembrolizumab, an anti-PD-1 mAb approved previously for treating a wide spectrum of tumors. Toripalimab, which binds the FG loop of PD-1, has 12-fold higher binding affinity to PD-1 than pembrolizumab and promotes significantly more Th1- and myeloid-derived inflammatory cytokine responses in healthy human PBMCs in vitro. In an ex vivo system employing dissociated tumor cells from treatment naïve non-small cell lung cancer patients, toripalimab induced several unique genes in IFN-γ and immune cell pathways, showed different kinetics of activation and significantly enhanced IFN-γ signature. Additionally, binding of toripalimab to PD-1 induced lower levels of SHP1 and SHP2 recruitment, the negative regulators of T cell activation, in Jurkat T cells ectopically expressing PD-1. Taken together, these data demonstrate that toripalimab is a potent anti-PD-1 antibody with high affinity PD-1 binding, strong functional attributes and demonstrated clinical activity that encourage its continued clinical investigation in several types of cancer.

Abstract C069: Toripalimab an anti-PD-1 antibody that demonstrates potent T cell activation and enhanced clinical efficacy irrespective of PD-L1 status

Abstract Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1) have revolutionized cancer treatment favoring prognostic benefits to a broad range of cancer patients. Currently, US Food and Drug Administration (FDA)–approved PD-1 monoclonal antibodies (mAbs) have demonstrated significant clinical benefit, particularly in patients with programmed cell death ligand 1 (PD-L1)-expressing tumors. Toripalimab is a PD-1 targeting humanized IgG4 mAb that is currently pending approval by the FDA as a first-line treatment for patients with recurrent or metastatic nasopharyngeal carcinoma (RM-NPC). In combination with chemotherapy, toripalimab demonstrated significant clinical efficacy irrespective of PD-L1 status in patients in three different clinical trials, namely JUPITER-02 (NPC), JUPITER-06 (esophageal squamous cell carcinoma [ESCC]) and CHOICE-01 (non-small cell lung cancer [NSCLC]). Here we investigated the molecular and functional characteristics of toripalimab and compared it to pembrolizumab, a PD-1 mAb that is approved in the largest number of indications in the PD-1 mAb class. In comparison to pembrolizumab, toripalimab demonstrated a 12-fold higher binding affinity to PD-1 and selectively induced higher Th1 and myeloid derived inflammatory cytokine responses in human peripheral blood mononuclear cells (PBMCs). Upon treating dissociated tumor cells from treatment naïve NSCLC patients, toripalimab demonstrated an enhanced expression of several unique genes in interferon gamma and immune cell pathways compared to pembrolizumab. The binding of toripalimab to PD-1 ectopically expressed in Jurkat T cells, recruited lower levels of SHP1 and SHP2, the negative regulators of T cell activation, compared to pembrolizumab. Overall, our study demonstrates that toripalimab is differentiated from pembrolizumab in terms of stronger PD-1 binding, more potent in-vitro T cell activation and lower agonistic potential. These characteristics of toripalimab present it as a next generation PD-1 checkpoint inhibitor with potential for favorable clinical outcomes in treating cancer patients irrespective of their PD-L1 status. Citation Format: Xiaoguang Wang, Sruthi Ravindranathan, Daniel Chin, Su-Yi Tseng, Scott Klakamp, Kate Widmann, Varun Kapoor, Vladimir Vexler, Patricia Keegan, Sheng Yao, Sanjay D Khare, Theresa LaVallee, Narendiran Rajasekaran. Toripalimab an anti-PD-1 antibody that demonstrates potent T cell activation and enhanced clinical efficacy irrespective of PD-L1 status [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C069.

Abstract 4436: A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1

Abstract It is well-established that T cells play a key role in the success of cancer immunotherapies. The goals of T cell-targeting therapies have been to restore the effector function of tumor-specific T cells that are either dysfunctional due to the immunosuppressive mechanisms in the tumor microenvironment, the lack of co-stimulatory signals, or negative regulation by expression of checkpoint molecules such as PD-1. E3 ligase Casitas B-Lineage Lymphoma Proto-Oncogene B (CBL-B) has recently gained attention as a master regulator of multiple immune-activation mechanisms. Inhibition of CBL-B enables the activation of antigen-specific T cells, and has been shown in syngeneic mouse tumor models to be efficacious as a cancer immunotherapy agent. CBL-B inhibition has also been shown to enhance and prolong the effects of anti-PD-1 antibody (Ab) therapy. However, genetic knockout of CBL-B in mice led to spontaneous autoimmunity characterized by auto-antibody production and infiltration of activated T cells and B cells into multiple organs that could result in tissue damage. In order to mitigate safety issues that could result from systemic CBL-B inhibition, we developed novel antibody-drug conjugates (ADCs) that target CBL-B inhibitors (CBL-Bi) to T cells via binding to PD-1. Using PD-1-expressing Jurkat NFAT reporter cells and mixed lymphocyte reaction assay, we demonstrated that our TPS2 approach enhanced the activation of T cells in vitro compared to treatment with anti-PD-1 Ab alone. Evaluation of pathways downstream of TCR activation in primary exhausted T cells showed increased Notch1, phospho-PLCγ2, and ZAP-70 accumulation following anti-PD-1/CBL-Bi treatment and is correlated with IFNγ induction. When autologous mature dendritic cells were co-cultured with dissociated tumor cells from melanoma patients, the highest magnitude of tumor-infiltrating lymphocyte activation was observed using delivery of CBL-Bi by anti-PD-1 ADC, compared to CBL-Bi alone, anti-PD-1 Ab alone, or combination treatment. The potential of anti-PD-1/CBL-Bi ADC for activation effector T cell function is also evident by increased intratumoral transcript levels of Granzyme B and Perforin following treatment in a humanized mouse model. In conclusion, we validated that the biological activity of CBL-Bi is retained after conjugation with an anti-PD-1 antibody. The use of anti-PD-1 Ab for developing the ADC not only targets the CBL-Bi payload to exhausted T cells but also blocks negative regulation of T cells via PD-1. Together, anti-PD-1/CBL-Bi ADC enhanced T cell activation in vitro as well as in vivo. Evaluation of safety and efficacy in animal tumor models is ongoing, to support further development of the TPS2 approach. Citation Format: Joanne Lim, Anna Skaletskaya, Uttapol Permpoon, Yeonjoon Kim, Shikha Saini, Khuloud Takrouri, Zinaida Ribkovskaia, Palash Bhar, Nathan Fishkin, Dong-Ki Choi, Ji Hyun Park, James Palacino, Peter U. Park. A novel antibody-enabled dual precision targeted protein stabilization (TPS2) that augments anti-tumor immune response by targeting CBL-B inhibitor to exhausted T cells while blocking checkpoint molecule, PD-1. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4436.

Abstract 602: Single cell evaluation of intratumoral B cells across solid tumor indications

Abstract The immune system is a critical component of the tumor microenvironment that has both tumor-promoting and tumor-eradicating activity based on the specific immune cell subtype and their activation status. T lymphocytes represent the largest lymphocyte population within the tumor, and numerous immunotherapies have been developed to specifically target this cellular subset. B cells are the next most abundant lymphocyte subset within the tumor microenvironment, and recent studies have identified crucial roles for intratumoral B cells in the production of tumor-reactive antibodies. Using human dissociated tumor cells (DTCs) samples, we have profiled intratumoral B cells to determine their frequency and subset breakdown across thirteen different solid tumor indications. Intratumoral B cells were prevalent in lung cancer and cancers of the gastrointestinal tract (esophageal, gastric, and colorectal), while they were rarer in cancers of the female reproductive tract (endometrial and ovarian). The transcriptional profile and B cell repertoire of the intratumoral B cell subsets were evaluated to provide a deeper understanding of the biology of these tumor-infiltrating lymphocyte subsets. Additionally, spatial transcriptomics were utilized to understand the spatiotemporal location of these subsets within the tumor microenvironment. These studies will provide critical insight for the development of the next generation of immunotherapies. Citation Format: Shawn P. Fahl, Kerri Colwell, Casey Smith, Cavin Ott, Alex Moore, Kayla Williams. Single cell evaluation of intratumoral B cells across solid tumor indications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 602.

Abstract 1617: TGFBR1 as a novel therapeutic target in adult granulosa cell tumors

Abstract Adult granulosa cell tumor (AGCT) is a subtype of sex-cord stromal tumors and accounts for ~5% of all ovarian neoplasms. Nearly 100% of AGCT cases are caused by an oncogenic point mutation in the Forkhead Box L2 (FOXL2) transcription factor. Weis-Banke et al. found this gain-of-function mutation (FOXL2C134W) allows FOXL2 to hijack the nuclear SMAD2/3/4 complex, the downstream effector of transforming growth factor- ß (TGF-ß), and redirect to novel transcription sites, inducing transcription of epithelial to mesenchymal transition (EMT) and other oncogenes. We hypothesized that FOXL2 mutant AGCT would be sensitive to TGF-ß inhibition. To test this hypothesis, we treated two GCT cell lines with TP-6379, an orally available, investigational small molecule kinase inhibitor of TGFBR1 that has been shown to block the phosphorylation and nuclear translocation of SMAD2 and SMAD3 in cells. TP-6379 was tested in the KGN cell line, derived from an AGCT patient and heterozygous for the FOXL2C134W mutation, and the COV434 cell line, derived from a juvenile GCT patient, which is FOXL2 wild type (WT). KGN cells (IC50 = 135 nM) were observed to be more than 70-fold more sensitive to TP-6379 treatment than COV434 (IC50 = >10,000 nM), after a 6- and 7- day treatment, respectively. KGN cells that were edited to remove the WT FOXL2 or both the WT and FOXL2C134W alleles were observed to be 1.6-fold more sensitive to TP-6379 and 22-fold less sensitive than the parental KGN cells. In vivo testing using KGN cells is ongoing. Viably cryopreserved dissociated tumor cells (DTCs) from two AGCT patients and one JGCT patient, which contain a mixture of tumor, immune, endothelial, and other stromal cells, were also tested in proliferation assays with TP-6379. All three ex vivo samples were positive for the FOXL2C134W mutation as detected by a qPCR genotyping assay and were sensitive to TP-6379 (IC50 = 555-1600 nM) after 6-day treatment. Xenograft models using these patient samples are currently under development. TGF-ß signaling is also a master regulator of the tumor microenvironment (TME) and immune evasion by modulating deposition of extracellular matrix and suppression of immune cells. We performed an immunophenotyping assay in tissue microarrays of thin-needle biopsy cores of multiple cancer types by looking at the distribution of CD8 T cells within tumor and stroma. AGCT showed the strongest excluded and desert phenotype among the tested cancer types, where CD8 T cells were confined to the stroma or absent entirely. TP-6379 treatment was observed to increase expression and reverse TGF-ß induced suppression of HLA class I in KGN cells. These data suggest that TGF-ß may play a significant role in the TME of AGCT. In conclusion, preclinical data shows inhibition of TGFß signaling with TP-6379 in FOXL2C134W mutant AGCT is active at blocking cell growth and may prove to be a potential therapy in this rare disease. Citation Format: Curtis A. Allred, Richard E. Heinz, Yuta Matsumura, Tetyana V. Forostyan, David Kircher, Salah Sommakia, Thomas Welte, Veena Vuttaradhi, Jason M. Foulks, Steven L. Warner, R Tyler Hillman. TGFBR1 as a novel therapeutic target in adult granulosa cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1617.

Abstract 681: ICVB-1042, an oncolytic adenovirus, infects and kills bladder, breast, and glioblastoma human dissociated tumor cells

Abstract ICVB-1042 is a novel E2F transcription factor-dependent oncolytic virus (OV) demonstrated to confer tumor selective replication, enhanced potency and tumor tropism. The lytic efficiency of ICVB-1042 was assessed in human primary dissociated tumor cells (DTCs) from bladder, breast, and glioblastoma (GBM) tumors and compared against indication specific OVs. Primary DTCs were analyzed on xCELLigence eSight instrument for simultaneous imaging and viability monitoring. ICVB-1042 and comparator OVs were added at multiplicities of infection (MOI) ranging from 1.2 - 30; plates were cultured for 10 days. Cell index measurements quantified viability. Imaging for a viral-encoded reporter gene (YPet) expression quantified viral infection. Supernatant from infected samples was analyzed by ddPCR to quantify viral genome replication. Efficacy of ICVB-1042 was assessed in a PDX model of subcutaneous GBM in Nude mice, with intratumoral administration of ICVB-1042 every 3 days for 3 doses. Table 1 reports cytolysis data. Bladder: ICVB-1042 infection showed complete lysis at MOI 30 and impressive lysis at lower MOIs. Comparator showed high levels of lysis only at MOI 30. Breast: ICVB-1042 showed efficient lysis at all MOIs; comparator did not reach >10% lysis in either donor. GBM: ICVB-1042 and comparator had similar lysis at MOI 30; ICVB-1042 showed efficient lysis at lower MOI. YPet imaging indicated ICVB-1042 infection spread more rapidly than comparators. ICVB-1042 produced more viral genomes via replication than comparators, particularly at high MOIs. ICVB-1042 showed significant tumor growth inhibition and increased overall survival compared to vehicle-treated animals. ICVB-1042 infection, replication, and cytolysis were evident across all diverse primary patient tumor types and multiple donor genotypes, with superior cytolysis compared to other indication specific OVs. ICVB-1042 maintains potent activity in vivo in patient GBM DCTs. Table 1. - Percentage OV-Mediated Cytolysis of Primary Human DTCs by Indication, Donor, and MOI Indication Bladder Breast GBM Donor MOI ICVB-1042 Comparator OV-1 (E1A promoter swap for tumor selectivity; hGMCSF transgene expression) MOI ICVB-1042 Comparator OV-2 (Ad11/Ad3 chimera; Generated by directed evolution for tumor selectivity) MOI ICVB-1042 Comparator OV-3 (E1A mutation for tumor selectivity; RDG-4C fiber mutation for enhanced tropism) Donor 1 30 100% 89.2% 30 83.4% 8% 30 100% 97.3% 6 100% 0% 6 90.7% 0% 7.5 100% 71.6% 1.2 68.6% 0% 1.2 62.3% 0% 1.88 86% 5.9% Donor 2 30 100% 94.4% 30 78% 6.9% 30 69.1% 53.9% 6 14.6% 0% 6 97.6% 9.6% 7.5 49.5% 0% 1.2 0% 0% 1.2 100% 8.8% 1.88 36.7% 0% Citation Format: Michael Pokrass, Peter Rosenthal, Joshua Messinger, Jessica Field, Heba Nowyhed. ICVB-1042, an oncolytic adenovirus, infects and kills bladder, breast, and glioblastoma human dissociated tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 681.

Tissue Factor (TF) Is Upregulated in Tumor-Bearing Mice Treated with Immune Checkpoint Inhibitor (ICI)

Introduction: Venous thromboembolism (VTE) is an important adverse event associated with immune checkpoint inhibitors (ICI) in cancer patients (12-15% annual rate). We have developed a murine model of venous thrombosis associated with ICI therapy (referred to as ICI-CAT). We found that mice bearing CT26 colon carcinoma treated with ICI developed larger thrombi following IVC occlusion compared with mice treated with IgG. ICI did not increase thrombus weight in mice without tumors. Tissue factor (TF) plays an important role in cancer-associated thrombosis (CAT) in patients treated with conventional therapies, and TF+ extracellular vesicles (EV) were associated with the development of VTE in pancreatic cancer. However, little is known about the impact of ICI on TF expression in patients treated with ICI. To address the underlying mechanism of ICI-associated thrombosis (IAT), we have examined the expression of TF in tumor and immune cells, as well as circulating EV, in our murine CT26 model. Methods: A syngeneic colon carcinoma model (CT26) in BALB/c mice was utilized to evaluate ICI-CAT following inferior vena cava ligation (stasis). Tumor tissue was analyzed for TF expression by immunoblotting using an anti-TF antibody (ab189483, Abcam). Tumor tissue was also dissociated, and TF-positive cells were assessed by flow cytometry analysis with CD45+ and CD45- gating using antibodies for CD11b, Ly6C, Ly6G (BioLegend), and TF (R&D). EV were isolated from platelet poor plasma from ICI or control IgG-treated mice by centrifugation (20,000 x g). Procoagulant activity of EV TF was measured using a chromogenic substrate to detect FXa generation following incubation of EV with FVIIa in the presence or absence of an anti-TF antibody. Results: Treatment of tumor-bearing mice with ICI markedly induced TF expression in tumors (Fig. A, Leftupper). Furthermore, levels of EV TF antigen (Fig. A, Left bottom) and EV TF activity (Fig. A, Right) were increased in mice treated with ICI. To explore the cellular origin of TF, we performed flow cytometry with dissociated tumor cells. A greater percentage of TF-positive cells in both the CD45+ (37.8%) and CD45- (74.6%) populations were detected in tumors from ICI treated mice compared to those treated with control IgG (27.9% and 49.8%, respectively), suggesting both tumor and tumor-infiltrating immune cells contribute to the larger thrombi observed in ICI-treated mice (Fig. B). Further analysis of CD45+ TF+ cells by CD11b gating (data not shown) revealed that a TF expression in a monocyte population (CD11b+ Ly6C+) was increased 3-fold by ICI (46.6%) compared to control IgG (14.2%) (Fig. C). We also found that TF expression was increased in cultured CT26 cells in response to interferon-g, but not in response to anti-PD-1 and anti-CTLA-4 antibodies, suggesting that ICIs do not directly upregulate TF and that more complex cellular interactions and inflammatory changes may be involved. Conclusions: ICI treatment of tumor-bearing mice results in increased tumor TF expression and EV TF expression. TF was also upregulated in immune cells in the tumor microenvironment after ICI treatment, including both tumor/stromal cells (CD45-), and tumor-associated monocytes and neutrophils (CD45+). These results suggest that multiple underlying mechanisms and diverse cellular expression underlie TF expression in this model, and that these processes many contribute to the larger thrombi observed in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Combination of Ribociclib with BET-Bromodomain and PI3K/mTOR Inhibitors for Medulloblastoma Treatment In Vitro and In Vivo.

Despite improvement in the treatment of medulloblastoma over the last years, numerous patients with MYC- and MYCN-driven tumors still fail current therapies. Medulloblastomas have an intact retinoblastoma protein RB, suggesting that CDK4/6 inhibition might represent a therapeutic strategy for which drug combination remains understudied. We conducted high-throughput drug combination screens in a Group3 (G3) medulloblastoma line using the CDK4/6 inhibitor (CDK4/6i) ribociclib at IC20, referred to as an anchor, and 87 oncology drugs approved by FDA or in clinical trials. Bromodomain and extra terminal (BET) and PI3K/mTOR inhibitors potentiated ribociclib inhibition of proliferation in an established cell line and freshly dissociated tumor cells from intracranial xenografts of G3 and Sonic hedgehog (SHH) medulloblastomas in vitro. A reverse combination screen using the BET inhibitor JQ1 as anchor, revealed CDK4/6i as the most potentiating drugs. In vivo, ribociclib showed single-agent activity in medulloblastoma models whereas JQ1 failed to show efficacy due to high clearance and insufficient free brain concentration. Despite in vitro synergy, combination of ribociclib with the PI3K/mTOR inhibitor paxalisib did not significantly improve the survival of G3 and SHH medulloblastoma-bearing mice compared with ribociclib alone. Molecular analysis of ribociclib and paxalisib-treated tumors revealed that E2F targets and PI3K/AKT/MTORC1 signaling genes were depleted, as expected. Importantly, in one untreated G3MB model HD-MB03, the PI3K/AKT/MTORC1 gene set was enriched in vitro compared with in vivo suggesting that the pathway displayed increased activity in vitro. Our data illustrate the difficulty in translating in vitro findings in vivo. See related article in Mol Cancer Ther (2022) 21(8):1306-1317.

Abstract 1686: Characterizing tumor heterogeneity through label-free, morphology-based live cell sorting

Abstract The complexity of cancer is compounded by the continuous emergence of malignant cell populations that drive tumor heterogeneity and challenges diagnostic and therapeutic advances. Methods to study cancer at the single cell level are important to better understand tumor heterogeneity. Isolation of tumor cells from tissue typically relies on targeting biomarkers overexpressed in tumor cells, such as EpCAM, resulting in cells that may express common markers with morphologic and molecular heterogeneity. We developed a platform termed Computational Sorting and Mapping of Single cells (COSMOS) that uses high-dimensional morphology analysis of single cells using artificial intelligence, microfluidics and high resolution bright-field imaging to identify and enrich target cells. We used COSMOS to train a deep convolutional neural network (ConvNet) classifier to identify and enrich malignant cells from non-small cell lung cancer (NSCLC) dissociated tumor cell (DTC) samples. The enriched population of NSCLC cells are label-free, unperturbed and viable, making them amenable to many downstream analysis methods but importantly also contain single cell high-dimensional morphology profiles. We verified enrichment of malignant cells by performing RNA and DNA analysis on the enriched sample. Single cell RNA-seq (scRNA-Seq) analysis shows high levels of EpCAM expression in sorted cells. Copy number variation (CNV) analysis demonstrated increased amplitude of deletion and amplification peaks relative to the pre-sorted DTC sample. Further, mutational analysis show increased allele frequency of mutations including P53 and KRAS in post-sorted compared to pre-sorted samples. Morphological heterogeneity within the malignant population is observed by UMAP analysis with the presence of multiple clusters of morphologically unique tumor cell populations detected. We further trained the classifier to detect and enrich for each subpopulation; CNV, mutation, bulk RNASeq, and scRNA-Seq analysis revealed molecular differences between the morphology subgroups. Further work is planned to evaluate the link between the morphological, molecular and functional characteristics of each subpopulation. We demonstrate a platform that can enrich malignant cells based on morphology, yielding a population of cells that are label-free, viable and unperturbed, making them compatible with downstream molecular assays commonly used to study heterogeneity. Additionally the cells can be morphologically profiled at the single cell level yielding a high-dimensional morphological profile that reveals heterogeneous populations of cells that can be further characterized by molecular analysis and could offer a new dimension to understand heterogeneity and biomarker discovery. Citation Format: Andreja Jovic, Kiran Saini, Michael Phelan, Ryan Chow, Simo Zhang, Chassidy Johnson, Nianzhen Li, Thomas J. Musci, Mahyar Salek, Maddison (Mahdokht) Masaeli. Characterizing tumor heterogeneity through label-free, morphology-based live cell sorting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1686.

Abstract 6057: Mechanisms mediating the radioresistance of human glioma cells growing in the murine olfactory bulb

Abstract We have previously reported (Int J Radiat Oncol Biol Phys. 107(1):194-201, 2020) that human GBM-stem like cells (GSCs) implanted into the right striatum of nude mice migrate/invade throughout the right hemisphere (RH) as well as into the olfactory bulb (OB). The highly invasive nature of the GSCs suggested a model system for evaluating the effects of specific brain microenvironments on glioma radioresponse and the potential for topographic heterogeneity in radioresistance. Subsequent analyses revealed that GSCs in the OB were significantly more radioresistant than those in the RH. Because the OB is highly enriched in neurons and given recent data suggesting that neuron/glioma interactions drive GBM growth, in this study we first defined contact between GSCs and neurons in the OB and RH using immunohistochemistry (IHC) and confocal microscopy. In the OB 56% of tumor cells were in direct contact with neurons as compared to 24% of tumor cells in the RH. Moreover, among the tumor cells in contact with neurons 25% had 2 or more neuronal contacts in the OB versus 2% in the RH. These data suggest a significantly greater level of glioma-neurons interaction in the OB. To further investigate the OB as a radioresistant niche, RNAseq was used to define the transcriptome of NSC11 GSCs located in the OB versus the RH. Of the greater than 15,000 genes evaluated, 1887 genes were differentially expressed between the 2 sites: 872 upregulated and 1015 down regulated in the OB versus the RH. Functional analyses (IPA and GSEA) indicated that among the genes overexpressed in the OB GSCs were a group associated with the cell cycle, specifically mitosis and the spindle assembly checkpoint (SAC). Comparison of the SAC genes overexpressed in the OB with GBM clinical outcomes (TCGA) showed a significant correlation with decreased patient survival. These analyses suggested that cell cycle regulation may play a role in the radioresistance of OB GSCs. Accordingly, radiation-induced cell cycle redistribution of GSCs in the OB and the RH was determined by IHC analysis of cell cycle phase markers and flow cytometry (propidium iodide) performed on dissociated tumor cells. Results showed a significantly higher percentage of cells in G2 in the OB at 24h and 48h after 10Gy, suggestive of prolonged G2 arrest. In addition, analysis of radiation-induced DNA DSBs using γH2AX and 53BP1 nuclear foci showed faster and more complete foci dispersal after 10Gy in the OB GSCs versus those in the RH, indicative of increased DSBs repair in the OB. These data suggest that GSC radioresistance in the OB is the result of a more effective DNA damage response. Moreover, results suggest that the murine OB may provide a model system for investigating GBM radioresistance as well as the potential role of glioma/neuron interactions. Citation Format: Charlotte M. Degorre, Ian C. Sutton, Stacey L. Lehman, Uma T. Shankavaram, Kevin A. Camphausen, Philip J. Tofilon. Mechanisms mediating the radioresistance of human glioma cells growing in the murine olfactory bulb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6057.

Abstract 3197: Blockade of regulatory signaling pathways reduces immunosuppression in the PDAC tumor microenvironment

Abstract Pancreatic cancer (PDAC) has the highest mortality rate of all major cancers, with few treatment options that provide long-term benefit to patients. Cancer associated fibroblasts (CAFs), the primary cellular component of the pancreatic tumor stroma, play a key role in the formation of an immunosuppressive tumor microenvironment (TME). A better understanding of the disease biology and cellular pathways that could be targeted for treatment of PDAC are needed. To investigate mechanisms driving immune suppression and pathways that might be targetable for treatment in PDAC, we used models where cross-talk between stromal and tumor cells could be evaluated. Three PDAC tumor cell lines were co-cultured (CC) with primary CAFs from multiple donors for 3 days, and secretion of numerous immunosuppressive cytokines, including IL6, IL8, IL10, CXCL12, was found to be upregulated in CC vs single culture (SC). IL6 and IL8 were shown to increase 2-38-fold in CC vs SC CAFs, and 1.5-20,000-fold vs SC PANCs, dependent upon cell line. By whole genome array, an upregulation of genes involved in NFkB, IL6, IL8, and AKT signaling was also observed (-log(p-value)>4) in CAF cells following CC. Multiple pathways have been implicated in regulating the immunosuppressive PDAC TME, including: CD73, a critical mediator of adenosine formation which is known to alter immune activity; and IL1, which is secreted by PDAC tumor cells, known to increase inflammation in the TME, and promote PDAC progression. The effects of CD73 inhibition and IL1R inhibition were evaluated to better characterize the downstream effects of these pathways on regulating tumor immunosuppression. Inhibition of CD73 in the CC reduced production of the immunosuppressive cytokines IL6, IL8, MCP1 CXCL12, IL1a, IL1b, IL10 (16-59%). Preliminary genomic studies indicated that inhibition of CD73 decreased genes involved in invasion, migration, metastasis, EMT and angiogenesis, as well as increasing genes involved in type 1 IFN activation, including OAS1, DDX60, IFIT1 and IRF7 (FC 1.5-2). Similarly, blockade of IL1R (which is highly expressed on PDAC CAFs and is the receptor for both IL1a and IL1b) reduced immune suppressive cytokine production from PDAC tumor conditioned media-stimulated CAFs, e.g. lowering secretion of IL6 and IL8 (4-20-fold) in a dose-dependent manner. Blocking IL1R in a PDAC dissociated tumor cell (DTC) model resulted in decreased secretion of CXCL1 and IL6 (60-75% over 3-6 days in culture), and an upregulation of IFNg (2-10-fold) in this culture by d3. These results suggest that a blockade of regulatory signaling pathways may be useful to reduce immune suppression and return the PDAC TME to a phenotype more amenable to immune checkpoint inhibition. Further characterization of these pathways may lead to new therapeutic combination approaches for pancreatic cancer. Citation Format: Lydia Greenlees, Li Cheng, Bilal Omar, Yelena Lazdun, Jixin Wang, Fernanda Pilataxi, Michael Kuziora, Zachary Cooper, Luis Vence, Katie Streicher. Blockade of regulatory signaling pathways reduces immunosuppression in the PDAC tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3197.

Abstract 183: Ex vivo 3D drug response profiling of XPDX-derived tumor cells for acceleration of preclinical drug development

Abstract Patient-Derived Xenografts (PDX) represent a versatile tool for preclinical drug development because they recapitulate many key features of the parent tumors, including molecular and histopathological profiles, tumor microenvironment, and tumor heterogeneity. The clinical relevance of PDX thus offer several advantages over other commonly used tools such as cell lines and genetically modified mice. Many large collections of PDX have been developed across a wide range of tumor types that profile the genetic diversity of each disease and the ability to bank PDX material allows repeated generation of mice for in vivo drug screens. As a result, PDX are used in several parts of the drug development pipeline, including early cancer biology studies, biomarker development, evaluation of therapeutic efficacy, and assessing/overcoming drug resistance. However, there remain drawbacks to this approach, most notably the large number of mice required for comprehensive drug screens and the associated time and costs. One approach to streamline PDX model selection and in vivo study execution would be to predict in vivo drug responses by assessing responses to the same drugs in an ex vivo platform utilizing PDX-derived dissociated tumor cells. KIYATEC’s KIYA-PREDICT™ PDX assay is a 3D spheroid-based ex vivo platform that has been used to screen a wide range of drugs and tumor types to predict drug responses in primary patient-derived tumors and PDX-derived tumors, including several PDX from XenoSTART’s extensive library of XPDX models. Here, we dissociated XPDX-derived tumors from a panel of 20 breast, ovarian, and lung cancer models provided by XenoSTART and evaluated their ex vivo responses to a panel of chemotherapy agents in our 3D KIYA-PREDICT™ assay. After exposure to drugs for 3-7 days, viability was assessed and both IC50s and percent survival values were calculated. The percent survival was then compared to in vivo drug response data provided by XenoSTART, and correlations between in vivo and ex vivo data were assessed. Drug responses were highly correlative between ex vivo and in vivo models, including the ability to recapitulate palbociclib resistance and platinum and taxane response. These results indicate that the KIYA-PREDICT™ PDX assay is a valuable tool to incorporate into drug development pipelines to accelerate the screening of new drug compounds on a wide range of clinically relevant samples and to guide selection of PDX models for in vivo studies. Citation Format: Aaron L. Carlson, Ashley K. Elrod, Natalie A. Williams, Alyssa D. Moriarty, Michael J. Wick, Teresa M. DesRochers. Ex vivo 3D drug response profiling of XPDX-derived tumor cells for acceleration of preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 183.

Low Colorectal Tumor Removal by E-Cadherin Destruction-Enabled Tumor Cell Dissociation.

Treatments for low colorectal cancer (CRC) remain a great challenge due to the heavy physical and psychological burdens of colostomy, strong drug toxicity in chemotherapy, and myelosuppression-/chemoradiation-related gastrointestinal symptoms. In this study, a highly biosafe and effective tumor cell dissociation-based low CRC treatment modality has been verified on both PDOs in vitro and colorectal tumor models in vivo. Notably, controllable EDTA release at the tumor sites was achieved by the LDH degradation in response to a slightly acidic microenvironment of low CRC tumors. Resultantly, the intratumoral E-cadherin for intercellular junctions of low CRC tumors was effectively destroyed via Ca2+ depletion by released EDTA from the interlayers, initiating remarkable tumor cell dissociation and resultant tumor disaggregation/removal via defecation. Dissociated tumor cells were prevailingly enveloped by LDH/EDTA, which prevented them from readhering to adjacent tissues, providing an unprecedented, efficient and safe therapeutic modality for low CRC, which will benefit patients suffering low CRC.

Abstract P5-06-08: Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis

Abstract Background: Metastatic disease is the foremost cause of breast cancer (BC) related mortality in women. One of the crucial challenges in reducing metastasis-related mortality is in identifying and understanding why certain BCs metastasize and recur. Although cancer cell-intrinsic factors are a key determinant in BC metastasis, host-intrinsic factors like the cells of the tumor microenvironment (TME), such as cancer-associated fibroblasts (CAFs) are what might be driving certain BCs to metastasize and recur. We recently showed that CAFs from the primary (TME) enter into circulation as cCAFs, form heterotypic clusters with tumor cells, and arrive at metastatic sites. This interaction between CAFs and tumor cells is crucial in furthering the metastatic cascade. However, the functional heterogeneity of the different CAF phenotypes and how that impacts BC metastasis is unknown. Stromal derived factor-1 (SDF-1/CXCL12) is an important chemokine that is known to be involved in promoting tumor cell invasion and metastasis. In this study, we examine the functional differences between CAF isolated from different molecular subtypes of BC. Furthermore, we elucidate the role of CAF secreted SDF-1 as being one of the mechanisms by which a subset of CAF cells permanently reprograms poorly-metastatic MCF-7 cells to augment EMT-driven genes and become metastatic in vivo. Methods: We used primary CAF cell lines derived from BCs of different molecular subtypes and examined their global gene expression profiles using RNAseq. We used NSG mice xenografted with MCF-7 BC cells and primary CAF cells to evaluate the contribution of two different CAF phenotypes in promoting BC progression and metastasis. We developed dissociated tumor cell lines from CAF co-injected MCF-7 xenografts to determine the molecular changes that CAFs impart to MCF-7 cells in vivo. Using limiting dilution tumor xenograft assays and gene expression profiles we examined the molecular pathways, BC stemness markers, and tumor-initiating capacity of the dissociated tumor cell lines. By long-term SDF-1 treatment of MCF-7 cells in vitro and using these cells in various RNASeq, in vitro, and in vivo assays we determined the role of SDF-1 in altering the phenotype of MCF-7 cells. Results: We found that CAFs from different molecular subtypes have a differential effect on tumor initiation, progression, and metastasis in xenograft assays. We also found that a subset of CAFs has the ability to make the poorly-metastatic MCF-7 cells metastatic in vivo. We also found that this subset of CAF cells has a higher expression of SDF-1 and the tumors formed with the co-injection of MCF-7 cells with this subset of CAF cells are enriched in cancer stem cell-like metastasis initiating cells. We also found that upon serial transplant, the tumors formed by CAF-reprogramed MCF-7 cells have a gene expression profile that shows enrichment for EMT-driven genes and these tumors are robustly metastatic. Furthermore, we found that the SDF-1/CXCR4 axis is one of the critical mechanisms by which this subset of CAF cells permanently reprograms the phenotype of the MCF-7 cells. Conclusions: CAFs are a highly heterogeneous population of cells in the breast TME. This study examines the contribution of the functional heterogeneity of the CAF cells in BC metastasis. We have found that a unique subset of CAF cells has the ability to reprogram a poorly metastatic BC cell to not only become metastatic but induce a permanent EMT-driven phenotypic shift in the BC cells. SDF-1 may be one of the mechanisms that drive this CAF-induced change and understanding the CAF functional landscape and its role in BC metastasis might be crucial to developing new therapeutic modalities to abrogate BC metastasis. Citation Format: Utsav Sharma, Jun Sun, Kelsie Medina-Saenz, Susan Bare, Philip Miller, Manuel Picon-Ruiz, Joyce Slingerland, Dorraya El-Ashry, Marc Lippman. Triple-negative breast cancer CAFs induce a metastatic phenotype in MCF-7 cells via the SDF-1/CXCR4 axis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-06-08.

Evaluation of cancer immunotherapy using mini-tumor chips.

Rationale: Predicting tumor responses to adjuvant therapies can potentially help guide treatment decisions and improve patient survival. Currently, tumor pathology, histology, and molecular profiles are being integrated into personalized profiles to guide therapeutic decisions. However, it remains a grand challenge to evaluate tumor responses to immunotherapy for personalized medicine.Methods: We present a microfluidics-based mini-tumor chip approach to predict tumor responses to cancer immunotherapy in a preclinical model. By uniformly infusing dissociated tumor cells into isolated microfluidic well-arrays, 960 mini-tumors could be uniformly generated on-chip, with each well representing the ex vivo tumor niche that preserves the original tumor cell composition and dynamic cell-cell interactions and autocrine/paracrine cytokines.Results: By incorporating time-lapse live-cell imaging, our mini-tumor chip allows the investigation of dynamic immune-tumor interactions as well as their responses to cancer immunotherapy (e.g., anti-PD1 treatment) in parallel within 36 hours. Additionally, by establishing orthotopic breast tumor models with constitutive differential PD-L1 expression levels, we showed that the on-chip interrogation of the primary tumor's responses to anti-PD1 as early as 10 days post tumor inoculation could predict the in vivo tumors' responses to anti-PD1 at the endpoint of day 24. We also demonstrated the application of this mini-tumor chip to interrogate on-chip responses of primary tumor cells isolated from primary human breast and renal tumor tissues.Conclusions: Our approach provides a simple, quick-turnaround solution to measure tumor responses to cancer immunotherapy.

Abstract 2241: Profiling tumor-infiltrating immune cells using an augmented transcriptome

Abstract Due to the complexity of tumor-immune interactions, comprehensive profiling of both the tumor and tumor microenvironment (TME) can help further our understanding of tumor progression and response to treatment. One significant application is quantification of the immune infiltrate, which has the potential to characterize response to checkpoint blockade therapy. While common experimental approaches exist to profile tumor-infiltrating immune cells, they can have significant practical limitations. Instead, RNA sequencing can be used to comprehensively profile the immune composition of the TME in a scalable way. To address this, we have developed an approach to quantify eight immune cell types in tumor samples, and compare it to quantification by orthogonal methods. This module adds to the ImmunoID NeXT Platform®, an augmented, immuno-oncology-optimized exome/transcriptome platform designed to provide comprehensive information regarding the tumor and TME from a single FFPE tumor sample. To generate our reference data, we profiled the transcriptomes of eight purified immune cell types using ImmunoID NeXT™. Then, we utilized multiple methodologies and sample types to generate orthogonal quantification of immune cells for testing. These include creating in vitro cell mixtures and profiling by flow cytometry, profiling healthy donor PBMCs with cytometry by time of flight (CyTOF), profiling of dissociated tumor cells (DTCs) with FACS, and immunofluorescence (IF) characterization of FFPE tumor samples. We also used ImmunoID NeXT to profile the immune infiltrate of over 800 tumor samples across 13 cancer types. We utilized the transcriptomes of the purified immune cells to develop reference expression signatures specific for each cell type. Then, we compared ImmunoID NeXT's transcriptome-based approach to the orthogonal quantification from the variety of samples. We observed a strong concordance of the quantification from our transcriptome-based approach to the flow cytometry results of the cell mixtures, demonstrating accuracy in purified immune cell populations. We also observed strong concordance to CyTOF results from healthy donor PBMC samples, showing accuracy in real samples with diverse immune populations. Further, we demonstrated strong concordance of our methodology to FACS results in DTCs and IF in FFPE tumor samples, ensuring that our approach is able to profile the immune composition in various tumor samples. Finally, we highlight the diversity of immune populations across cancer types by applying ImmunoID NeXT to over 800 tumor samples. RNA sequencing can be used as a scalable approach to profile the immune infiltrate in tumors. Such analysis can add to our understanding of the tumor-immune interaction, including studies of response to immunotherapy. We show that the ImmunoID NeXT Platform is able to accurately evaluate the composition of tumor-infiltrating immune cells. Citation Format: Eric Levy, Pamela Milani, Fabio Navarro, Charles W. Abbott, Robert Power, John West, John Lyle, Sean M. Boyle, Richard Chen. Profiling tumor-infiltrating immune cells using an augmented transcriptome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2241.