Tumor-stroma Interactions Research Articles

Proteomic analysis of non-muscle invasive and muscle invasive bladder cancer highlights distinct subgroups with metabolic, matrisomal, and immune hallmarks and emphasizes importance of the stromal compartment.

We present the proteomic profiling of 79 bladder cancers, including treatment-naïve non-muscle-invasive bladder cancer (NMIBC, n = 17), muscle-invasive bladder cancer (MIBC, n = 51), and neoadjuvant-treated MIBC (n = 11). Proteins were extracted from formalin-fixed, paraffin-embedded samples and analyzed using data-independent acquisition, yielding >8,000 quantified proteins. MIBC, compared to NMIBC, shows an extracellular matrix (ECM) and immune response signature as well as alteration of the metabolic proteome together with concomitant depletion of proteins involved in cell-cell adhesion and lipid metabolism. Neoadjuvant treatment did not consistently impact the proteome of the residual tumor mass. NMIBC presents two proteomic subgroups that correlate with histological grade and feature signatures of cell adhesion or lipid/DNA metabolism. Treatment-naïve MIBC presents three proteomic subgroups with resemblance to the basal-squamous, stroma-rich, or luminal subtypes and signatures of metabolism, immune functionality, or ECM. The metabolic subgroup presents an immune-depleted microenvironment, whereas the ECM and immune subgroups are enriched for markers of M2-like tumor-associated macrophages and dendritic cells. Markers for natural killer cells are exclusive for the ECM subgroup, and markers for cytotoxic T cells are a hallmark of the immune subgroup. Endogenous proteolysis is increased in MIBC alongside upregulation of matrix metalloproteases, including MMP-14. Genomic panel sequencing yielded the prototypical profile of prevalent FGRF3 alterations in NMIBC and TP53 alterations in MIBC. Tumor-stroma interactions of MIBC were investigated by proteomic analysis of patient-derived xenografts, highlighting specific tumor and stroma contributions to the matrisome and tumor-induced stromal proteome phenotypes. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Abstract C034: Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors

Abstract Although various effective anti-cancer treatments have become available over the last decades, resistance to all available therapies remains the major cause of death of cancer patients with disseminated tumors. Striking examples are patients with triple-negative breast cancer (TNBC), which are frequently defective in the repair of DNA double strand breaks, e.g. due to loss of BRCA1 function. Because of this defect, the patients initially respond very well to DNA damage-inducing chemotherapy. Unfortunately, disseminated tumors are usually not eradicated and resistant tumor cells are eventually selected from residual primary or metastatic tumor sites. It is therefore crucial to understand the molecular mechanisms underlying the drug tolerance of the residual tumor cells. To study residual disease, we used the K14cre;Brca1 F/F ;p53 F/F (KB1P) mouse model for hereditary breast cancer, which provides the unique opportunity to explore and target those mechanisms in a fully immunocompetent model. The mammary tumors that spontaneously develop highly resemble their human counterparts, both morphologically and in their therapy response. For example, tumors shrink in response to poly(ADP-ribose) polymerase inhibition, platinum-based treatment or the commonly used doxorubicin, docetaxel and cyclophosphamide (TAC) combination therapy. But despite repeated drug sensitivity, the KB1P mammary tumors are not eradicated, not even by a frequent dosing schedule. By combining single-cell RNA sequencing, spatial transcriptomics and imaging mass cytometry, we dissected the intratumoral heterogeneity and alterations in the tumor microenvironment of residual disease. We identified specific subpopulations of tumor cells that have a survival benefit after treatment, and these occurred together with an altered microenvironment characterized by a highly reactive stroma infiltrated with both anti-inflammatory and pro-tumoral immune cells. Interestingly, those structural and transcriptional changes are reversed in the relapsed tumors, highlighting the plasticity of drug tolerance. To investigate the mechanisms of the altered tumor-stroma interactions that are relevant for drug tolerance, we have designed a custom-made CRISPR/Cas9 library based on differential gene expression of the residual tumor cells. This library enables us to functionally test relevant mechanisms of drug tolerance in vivo and we expect that these analyses will provide useful insights into the tumor-stroma interactions that contribute to residual disease. Taken together, our detailed analyses demonstrate the substantial remodeling of tumor cells in their microenvironment upon treatment. To develop new therapeutic approaches to eradicate drug-tolerant tumor cells and thereby circumvent tumor relapse, it is essential to better understand the heterogeneous cellular composition of residual tumors in its spatial context. With this project, we hope to provide comprehensive data to develop better therapeutic strategies that target the tumor-stroma interaction and eradicate residual tumors. Citation Format: Morgane Decollogny, Demeter Túrós, Astrid Chanfon, Myriam Siffert, Ismar Klebic, Sven Rottenberg. Investigating residual disease in its spatial context in BRCA1 p53-deficient mammary tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C034.

Abstract IA020: Deciphering the mechanisms of neutrophil immune response in bone metastatic prostate cancer

Abstract Metastatic disease of prostate cancer (PCa) is currently incurable. Bone is the most common tissue site for prostate cancer metastasis and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone marrow neutrophils inhibit bone metastatic PCa growth, however metastatic PCa becomes resistant to neutrophil response and progresses within the bone compartment. It is unclear how these findings translate to a clinical setting. Further, the mechanisms associated with tumor resistance to neutrophil immune response remain unknown and presents a new avenue for therapeutic intervention. Thus, the goals of this study were: 1) to define the impact of metastatic PCa on neutrophil function throughout prostate cancer progression and 2) to determine the potential of neutrophils as predictive biomarkers of PCa disease progression. To do this, peripheral blood polymorphonuclear neutrophils (PMNs) were collected from 4 patient cohorts (localized PCa, metastatic hormone-sensitive PCa (mHSPC), metastatic castration-resistant (mCRPC) and healthy men) and PMN molecular and functional properties assessed, including: cancer cell cytotoxicity and cell surface markers. There was a significant decrease with disease progression in PMN expression of CD10, a marker for differentiation, while in tandem, there was an increase in pro-inflammatory marker, CD88 (complement receptor) in mCRPC men compared to mHSPC. PMNs from all disease stages were equally cytotoxic to human PCa cell lines (LNCaP, C42B, and PC3). However, second generation androgen deprivation therapy (ADT) suppressed patient PMN cytotoxicity against mCRPC cells (C42B, PC3). This suppression was due to androgen receptor (AR)-mediated regulation of the pleotropic and immune suppressant cytokine, transforming growth factor beta receptor I (TβRI). PMNs from mCRPC patients who received 2nd generation ADT, expressed significantly more TβRI than patients who only received 1st line ADT. Further, we found that PMNs in CRPC patient bone metastases express more TβRI than PMNs in matched liver metastases, which express little to no TβRI. Treatment of mouse bone marrow neutrophils with enzalutamide, increased neutrophil TβRI expression in a dose dependent manner and blocked cytotoxicity against PCa cells. Exogenous testosterone treatment in vivo, pharmacologic inhibition (using Repsox, a small molecule kinase inhibitor of TβRI) or genetic deletion (using conditional TβRI knockout mice) rescued enzalutamide-mediated neutrophil suppression and restored neutrophil cytotoxicity. These data collectively suggest that AR inhibition suppresses anti-tumor neutrophil responses via TβRI however, this study highlights the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa. Citation Format: Leah M Cook. Deciphering the mechanisms of neutrophil immune response in bone metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr IA020.

YKL40/Integrin β4 Axis Induced by the Interaction between Cancer Cells and Tumor-Associated Macrophages Is Involved in the Progression of High-Grade Serous Ovarian Carcinoma.

Macrophages in the tumor microenvironment, termed tumor-associated macrophages (TAMs), promote the progression of various cancer types. However, many mechanisms related to tumor-stromal interactions in epithelial ovarian cancer (EOC) progression remain unclear. High-grade serous ovarian carcinoma (HGSOC) is the most malignant EOC subtype. Herein, immunohistochemistry was performed on 65 HGSOC tissue samples, revealing that patients with a higher infiltration of CD68+, CD163+, and CD204+ macrophages had a poorer prognosis. We subsequently established an indirect co-culture system between macrophages and EOC cells, including HGSOC cells. The co-cultured macrophages showed increased expression of the TAM markers CD163 and CD204, and the co-cultured EOC cells exhibited enhanced proliferation, migration, and invasion. Cytokine array analysis revealed higher YKL40 secretion in the indirect co-culture system. The addition of YKL40 increased proliferation, migration, and invasion via extracellular signal-regulated kinase (Erk) signaling in EOC cells. The knockdown of integrin β4, one of the YKL40 receptors, suppressed YKL40-induced proliferation, migration, and invasion, as well as Erk phosphorylation in some EOC cells. Database analysis showed that high-level expression of YKL40 and integrin β4 correlated with a poor prognosis in patients with serous ovarian carcinoma. Therefore, the YKL40/integrin β4 axis may play a role in ovarian cancer progression.

Abstract A055: Multiomics profiling reveals druggable tumor-stroma interactions in ATM-deficient pancreatic cancer

Abstract The complex biology of pancreatic ductal adenocarcinoma (PDAC) remains a significant challenge and limits overcoming its dismal prognosis. The tumor microenvironment (TME), shaped by dynamic interactions between cancer-associated fibroblasts (CAFs), immune and tumor cells, plays a crucial role in PDAC pathogenesis. Uncharted field lies in the impact of specific gene mutations within the tumor epithelium on orchestrating distinct oncogenic TME patterns. Our investigation into homologous recombination deficiency (HRD), caused by mutations in genes like ATM, reveals its role in promoting PDAC aggressiveness and triggering a pronounced desmoplastic reaction, suggesting a unique TME programing. Through single-cell-resolved and multiomics analyses of murine ATM and/or P53-depleted PDACs, and validation in human cases, we unveil transcriptional, translational, and secretory regulatory events, shed light on the intricate interplay between tumor genetics and PDAC microenvironment and pave the way for the elaboration of novel personalized therapies. Murine tumor analysis using single-nucleus multiomics RNA and ATAC sequencing, alongside histological validation in human tissues, revealed specific enrichment of myCAFs in ATM-deficient tumors and reduced populations of T cells and macrophages. Molecular signatures and cell-cell communication inference uncovered ATM-specific interactions within the TME, highlighting TGF-β signaling as pivotal in mediating dialog between CAFs and an aggressive EMT-like tumor cell subset prevalent in ATM-depleted tumors. Additionally, distinct communication patterns were observed between CAFs and immune cells, associated with specific epigenetic and molecular profiles. Mechanistically, transcriptomics, secretomics and proteomics investigations confirmed increased TGFβ release by ATM-deficient PDAC cells and identified a reactive oxygen species (ROS)-mediated mechanism affecting tumor cell behavior and interactions with CAFs. Consistently, a combinatorial therapy targeting TGF-β-mediated tumor-stroma dialog reversed cancer-promoting TME remodeling and exacerbated FOLFIRINOX cytotoxic effects, in vivo, exclusively in ATM-deficient HRD PDACs. Dissecting the biology of ATM-deficient HRD malignant cells, our study unveils their pivotal role in orchestrating oncogenic communication networks with CAFs and immune cells, along with specific tumor promoting stromal reprogramming. Our findings paint a comprehensive picture of how distinct tumor-stroma interactions reshape the TME, steering it towards a cancer-promoting outcome and shed light on the potential to exploit genotype-specific vulnerabilities via tailored tumor-stroma interference strategies. Citation Format: Elodie Roger, Hannah M. Mummey, Eleni Zimmer, Julia Ragg, Yuna Lee, Kyle J. Gaulton, Alica K. Beutel, Christopher J. Halbrook, Lukas Perkhofer, Johann Gout, Alexander Kleger. Multiomics profiling reveals druggable tumor-stroma interactions in ATM-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A055.

Multicompartmentalized Microvascularized Tumor-on-a-Chip to Study Tumor-Stroma Interactions and Drug Resistance in Ovarian Cancer

IntroductionThe majority of ovarian cancer (OC) patients receiving standard of care chemotherapy develop chemoresistance within 5 years. The tumor microenvironment (TME) is a dynamic and influential player in disease progression and therapeutic response. However, there is a lack of models that allow us to elucidate the compartmentalized nature of TME in a controllable, yet physiologically relevant manner and its critical role in modulating drug resistance.MethodsWe developed a 3D microvascularized multiniche tumor-on-a-chip formed by five chambers (central cancer chamber, flanked by two lateral stromal chambers and two external circulation chambers) to recapitulate OC-TME compartmentalization and study its influence on drug resistance. Stromal chambers included endothelial cells alone or cocultured with normal fibroblasts or cancer-associated fibroblasts (CAF).ResultsThe tumor-on-a-chip recapitulated spatial TME compartmentalization including vessel-like structure, stromal-mediated extracellular matrix (ECM) remodeling, generation of oxygen gradients, and delayed drug diffusion/penetration from the circulation chamber towards the cancer chamber. The cancer chamber mimicked metastasis-like migration and increased drug resistance to carboplatin/paclitaxel treatment in the presence of CAF when compared to normal fibroblasts. CAF-mediated drug resistance was rescued by ECM targeted therapy. Critically, these results demonstrate that cellular crosstalk recreation and spatial organization through compartmentalization are essential to determining the effect of the compartmentalized OC-TME on drug resistance.ConclusionsOur results present a functionally characterized microvascularized multiniche tumor-on-a-chip able to recapitulate TME compartmentalization influencing drug resistance. This technology holds the potential to guide the design of more effective and targeted therapeutic strategies to overcome chemoresistance in OC.

Targeting the cancer cells and cancer-associated fibroblasts with next-generation FGFR inhibitors in prostate cancer co-culture models.

Inhibition of androgen receptor (AR) signaling is the main treatment strategy in advanced prostate cancer (PCa). A subset of castration resistant prostate cancer (CRPC) bypasses the AR blockade by increased fibroblast growth factor receptor (FGFR) signaling. The first- and second-generation, non-covalent FGFR inhibitors (FGFRis) have largely failed in the clinical trials against PCa. In this study, we tested the drug sensitivity of LNCaP, VCaP, and CWR-R1PCa cell lines to second-generation, covalent FGFRis (FIIN1, FIIN2) and a novel FGFR downstream molecule inhibitor (FRS2αi). 2D and 3D mono- and co-cultures of cancer cells, and cancer-associated fibroblasts (CAFs) were used to mimic tumor-stroma interactions in the extracellular matrix (ECM). The treatment responses of the FGFR signaling molecules, the viability and proliferation of cancer cells, and CAFs were determined through immunoblotting, migration assay, cell viability assay, and real-time imaging. Immunofluorescent and confocal microscopy images of control and treated cultures of cancer cells and CAFs, and their morphometric data were deduced. The FGFRis were more effective in mono-cultures of the cancer cells compared with co-cultures with CAFs. The FRS2αi was specifically effective in co-cultures with CAFs but was not cytotoxic to CAF mono-cultures as in the case of FIIN1 and FIIN2. At the molecular level, FRS2αi decreased p-FRS2α, p-ERK1/2, and activated apoptosis as monitored by cleaved caspase-3 activity in a concentration-dependent manner in the co-cultures. We observed no synergistic drug efficacy in the combination treatment of the FGFRi with ARi, enzalutamide, and darolutamide. The FRS2αi treatment led to a decrease in proliferation of cancer cell clusters in co-cultures as indicated by their reduced size and Ki67 expression. CAFs exert a protective effect on cancer cells and should be included in the invitro models to make them physiologically more relevant in screening and testing of FGFRis. The FRS2αi was the most potent agent in reducing the viability and proliferation of the 3D organotypic co-cultures, mainly by disrupting the contact between CAFs and cancer cell clusters. The next-generation FGFRi, FRS2αi, may be a better alternative treatment option for overcoming ARi treatment resistance in advanced PCa.

Molecular Alterations Associated with Histologically Overt Stromal Response in Patients with Prostate Cancer.

Prostate cancer has substantial heterogeneity in clinical outcomes and therapeutic responses, posing challenges in predicting disease progression and tailoring treatment strategies. Recent studies have highlighted the potential prognostic value of evaluating the tumor microenvironment, including the presence of a histologically overt stromal response (HOST-response) characterized by peri-glandular stromal changes and architectural distortions. This retrospective study examined patient records from The Cancer Genome Atlas database to identify genomic alterations associated with the HOST-response in prostate cancer. Among 348 patients who underwent radical prostatectomy, 160 (45.98%) were identified as having a HOST-response. A gene expression analysis revealed 1263 genes with significantly higher expression in patients with a HOST-response. A protein-protein interaction network analysis identified seven hub genes (KIF2C, CENPA, CDC20, UBE2C, ESPL1, KIF23, and PLK1) highly interconnected in the network. A functional enrichment analysis revealed alterations in the cell division, cytoskeletal organization, cytokinesis, and interleukin-16 signaling pathways in patients with a HOST-response, suggesting dysregulated proliferation and inflammation. The distinct molecular signature associated with the HOST-response provides insights into the tumor-stroma interactions driving adverse outcomes and potential targets for tailored therapeutic interventions in this subset of patients with prostate cancer.

Targeting CERS6-AS1/FGFR1 axis as synthetic vulnerability to constrain stromal cells supported proliferation in Mantle cell lymphoma.

The interaction between stromal and tumor cells in tumor microenvironment is a crucial factor in Mantle cell lymphoma (MCL) progression and therapy resistance. We have identified a long non-coding RNA, CERS6-AS1, upregulated in MCL and associated with poor overall survival. CERS6-AS1 expression was elevated in primary MCL within stromal microenvironment and in a subset of MCL cells adhered to stromal layer. These stromal-adhered MCL-subsets exhibited cancer stem cell signatures than suspension counterparts. Mechanistically, we found that downregulating CERS6-AS1 in MCL reduced Fibroblast Growth Factor Receptor-1 (FGFR1), expression attributed to loss of its interaction with RNA-binding protein nucleolin. In addition, using in-silico approach, we have discovered a direct interaction between nucleolin and 5'UTR of FGFR1, thereby regulating FGFR1 transcript stability. We discovered a positive association of CERS6-AS1 with cancer stem cell signatures, and Wnt signaling. Building on these, we explored potential therapeutic strategies where combining nucleolin-targeting agent with FGFR1 inhibition significantly contributed to reversing cancer stem cell signatures and abrogated primary MCL cell growth on stromal layer. These findings provide mechanistic insights into regulatory network involving CERS6-AS1, nucleolin, and FGFR1 axis-associated crosstalk between tumor cells and stromal cell interaction and highlights therapeutic potential of targeting a non-coding RNA in MCL.

Osteopontin: A Key Multifaceted Regulator in Tumor Progression and Immunomodulation.

The tumor microenvironment (TME) is composed of various cellular components such as tumor cells, stromal cells including fibroblasts, adipocytes, mast cells, lymphatic vascular cells and infiltrating immune cells, macrophages, dendritic cells and lymphocytes. The intricate interplay between these cells influences tumor growth, metastasis and therapy failure. Significant advancements in breast cancer therapy have resulted in a substantial decrease in mortality. However, existing cancer treatments frequently result in toxicity and nonspecific side effects. Therefore, improving targeted drug delivery and increasing the efficacy of drugs is crucial for enhancing treatment outcome and reducing the burden of toxicity. In this review, we have provided an overview of how tumor and stroma-derived osteopontin (OPN) plays a key role in regulating the oncogenic potential of various cancers including breast. Next, we dissected the signaling network by which OPN regulates tumor progression through interaction with selective integrins and CD44 receptors. This review addresses the latest advancements in the roles of splice variants of OPN in cancer progression and OPN-mediated tumor-stromal interaction, EMT, CSC enhancement, immunomodulation, metastasis, chemoresistance and metabolic reprogramming, and further suggests that OPN might be a potential therapeutic target and prognostic biomarker for the evolving landscape of cancer management.

Effect of Bioactive Black Phosphorus Nanomaterials on Cancer-Associated Fibroblast Heterogeneity in Pancreatic Cancer.

Tumor-stromal interactions and stromal heterogeneity in the tumor microenvironment are critical factors that influence the progression, metastasis, and chemoresistance of pancreatic ductal adenocarcinoma (PDAC). Here, we used spatial transcriptome technology to profile the gene expression landscape of primary PDAC and liver metastatic PDAC after bioactive black phosphorus nanomaterial (bioactive BP) treatment using a murine model of PDAC (LSL-KrasG12D/+; LSL-Trp53R172H/+; and Pdx-1-Cre mice). Bioinformatic and biochemical analyses showed that bioactive BP contributes to the tumor-stromal interplay by suppressing cancer-associated fibroblast (CAF) activation. Our results showed that bioactive BP contributes to CAF heterogeneity by decreasing the amount of inflammatory CAFs and myofibroblastic CAFs, two CAF subpopulations. Our study demonstrates the influence of bioactive BP on tumor-stromal interactions and CAF heterogeneity and suggests bioactive BP as a potential PDAC treatment.

Phase II trial of BXCL701 and pembrolizumab in patients with metastatic pancreatic ductal adenocarcinoma (EXPEL-PANC): Preliminary findings.

LBA4132 Background: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options and is thought to be a “cold” tumor that does not respond to immunotherapy, due to a tumor microenvironment (TME) consisting of a desmoplastic stroma and poor T cell infiltrate. BXCL701 is an oral synthetic dipeptide that competitively inhibits dipeptidyl peptidases DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). BXCL701 exerts antitumor activity via inhibition of DPP8/9, which is associated with induction of proinflammatory cytokines, as well as inhibition of FAP, which disrupts tumor-stromal interactions. Preclinical xenograft models demonstrate synergy between BXCL701 and PD-1 blockade, reducing tumor growth and promoting an increase in intratumoral CD4+ and CD8+ T cells, macrophages and NK cells, with induction of host-protective immunity. Methods: This is a phase II trial of BXCL701 in patients with metastatic PDAC (mPDAC) following progression on 1 line of treatment for advanced disease and amenable to serial biopsies. BXCL701 is administered at 0.2 mg PO BID days 1-7 and 0.3 mg BID days 8-14 during cycle 1 (21 days) followed by 0.3 mg BID days 1-14 every 21 days in all other cycles, given with pembrolizumab 200 mg IV every 21 days (all cycles). The primary objective is to determine the 18-week progression-free survival rate (PFS18weeks). We estimate that historical 2nd-line PFS18weeks is 30% or less; using a Simon’s two-stage (minimax) design, a type I error rate of 0.05 and power of 80% if the true rate is 50%, we will need 19 patients in stage 1 and 20 in stage 2 (39 total). There is a safety lead-in phase of 6 patients. We plan to enroll 43 patients to account for a predicted 10% drop out of unevaluable patients. Correlative pharmacodynamic studies include imaging mass cytometry to examine 36 markers of the PDAC TME in tissue biopsies, as well as blood-based analyses of KRAS circulating tumor DNA, circulating markers of fibrosis, and IL-6. Enrollment began in Q3 2023 (NCT05558982). Results: Six patients have enrolled, 3 women and 3 men, median age 57.5 (range 37-80). One patient was progression-free at 18 weeks and 1 patient had stable disease (SD) at 9 weeks, not yet evaluable for the 18-week landmark. Objective response rate is 16% and disease control rate is 50% (RECIST: 1 partial response, -41%, and 2 patients with SD, -18% and 0%). Three patients had significant reductions in CA19-9 from baseline (-100%, -73%, and -97%). Median PFS and overall survival have not been reached (NR, 95% CI 1.45 months-NR and 0.92 months-NR, respectively). There have been no serious treatment-related safety events. The safety lead-in will complete after the next patient completes the 6-week safety window (1 patient was unevaluable). Conclusions: BXCL701 plus pembrolizumab is well-tolerated and shows early signs of potential clinical activity in patients with mPDAC refractory to chemotherapy. Clinical trial information: NCT05558982 .

Multicompartmentalized microvascularized tumor-on-a-chip to study tumor-stroma interactions and drug resistance in ovarian cancer.

The majority of ovarian cancer (OC) patients receiving standard of care chemotherapy develop chemoresistance within 5 years. The tumor microenvironment (TME) is a dynamic and influential player in disease progression and therapeutic response. However, there is a lack of models that allow us to elucidate the compartmentalized nature of TME in a controllable, yet physiologically relevant manner and its critical role in modulating drug resistance. We developed a 3D microvascularized multiniche tumor-on-a-chip formed by five chambers (central cancer chamber, flanked by two lateral stromal chambers and two external circulation chambers) to recapitulate OC-TME compartmentalization and study its influence on drug resistance. Stromal chambers included endothelial cells alone or cocultured with normal fibroblasts or cancer-associated fibroblasts (CAF). The tumor-on-a-chip recapitulated spatial TME compartmentalization including vessel-like structure, stromal-mediated extracellular matrix (ECM) remodeling, generation of oxygen gradients, and delayed drug diffusion/penetration from the circulation chamber towards the cancer chamber. The cancer chamber mimicked metastasis-like migration and increased drug resistance to carboplatin/paclitaxel treatment in the presence of CAF when compared to normal fibroblasts. CAF-mediated drug resistance was rescued by ECM targeted therapy. Critically, these results demonstrate that cellular crosstalk recreation and spatial organization through compartmentalization are essential to determining the effect of the compartmentalized OC-TME on drug resistance. Our results present a functionally characterized microvascularized multiniche tumor-on-a-chip able to recapitulate TME compartmentalization influencing drug resistance. This technology holds the potential to guide the design of more effective and targeted therapeutic strategies to overcome chemoresistance in OC.

Identification of Small-Molecule Inhibitors Targeting Different Signaling Pathways in Cancer-Associated Fibroblast Reprogramming under Tumor–Stroma Interaction

Cancer-associated fibroblasts (CAFs) interact reciprocally with tumor cells through various signaling pathways in many cancer types, including cutaneous squamous cell carcinoma. Among normal fibroblast subtypes, papillary fibroblasts (PFs) and reticular fibroblasts (RFs) respond distinctly to tumor cell signaling, eventuating the differentiation of RFs rather than PFs into CAFs. The regulation of subtype differentiation in fibroblasts remains poorly explored. In this study, we assessed the differences between PFs, RFs, and CAFs and examined the effects of small-molecule inhibitors targeting the TGFβ, phosphoinositide 3-kinase/protein kinase B/mTOR, and NOTCH pathways on the tumor-promoting property of CAFs and CAF reprogramming in 2-dimensional and 3-dimensional cultures. Blocking TGFβ and phosphoinositide 3-kinase strongly deactivated and concurrently induced a PF phenotype in RFs and CAFs. Three-dimensional coculturing of a cutaneous squamous cell carcinoma cell line MET2 with RFs or CAFs led to enhanced tumor invasion, RF-CAF transition, and cytokine production, which were further repressed by blocking TGFβ and phosphoinositide 3-kinase/mTOR pathways but not NOTCH pathway. In conclusion, the study identified biomarkers for PFs, RFs, and CAFs and displayed different effects of blocking key signaling pathways in CAFs and tumor cell-CAF interplay. These findings prompted a CAF-to-PF therapeutic strategy and provided perspectives of using included inhibitors in CAF-based cancer therapy.

Spatial transcriptomics analysis identifies a tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease

Leptomeningeal disease (LMD) remains a rapidly lethal complication for late-stage melanoma patients. Here, we characterize the tumor microenvironment of LMD and patient-matched extra-cranial metastases using spatial transcriptomics in a small number of clinical specimens (nine tissues from two patients) with extensive invitro and invivo validation. The spatial landscape of melanoma LMD is characterized by a lack of immune infiltration and instead exhibits a higher level of stromal involvement. The tumor-stroma interactions at the leptomeninges activate tumor-promoting signaling, mediated through upregulation of SERPINA3. The meningeal stroma is required for melanoma cells to survive in the cerebrospinal fluid (CSF) and promotes MAPK inhibitor resistance. Knocking down SERPINA3 or inhibiting the downstream IGR1R/PI3K/AKT axis results in tumor cell death and re-sensitization to MAPK-targeting therapy. Our data provide a spatial atlas of melanoma LMD, identify the tumor-promoting role of meningeal stroma, and demonstrate a mechanism for overcoming microenvironment-mediated drug resistance in LMD.

A bioprinted sea-and-island multicellular model for dissecting human pancreatic tumor-stroma reciprocity and adaptive metabolism

Pancreatic ductal adenocarcinoma (PDAC) presents a formidable clinical challenge due to its intricate microenvironment characterized by desmoplasia and complex tumor-stroma interactions. Conventional models hinder studying cellular crosstalk for therapeutic development. To recapitulate key features of PDAC masses, this study creates a novel sea-and-island PDAC tumor construct (s&i PTC). The s&i PTC consists of 3D-printed islands of human PDAC cells positioned within an interstitial extracellular matrix (ECM) populated by human cancer-associated fibroblasts (CAFs). This design closely mimics the in vivo desmoplastic architecture and nutrient-poor conditions. The model enables studying dynamic tumor-stroma crosstalk and signaling reciprocity, revealing both known and yet-to-be-discovered multicellular metabolic adaptations. Using the model, we discovered the orchestrated dynamic alterations of CAFs under nutrient stress, resembling critical in vivo human tumor niches, such as the secretion of pro-tumoral inflammatory factors. Additionally, nutrient scarcity induces dynamic alterations in the ECM composition and exacerbates poor cancer cell differentiation—features well-established in PDAC progression. Proteomic analysis unveiled the enrichment of proteins associated with aggressive tumor behavior and ECM remodeling in response to poor nutritional conditions, mimicking the metabolic stresses experienced by avascular pancreatic tumor cores. Importantly, the model's relevance to patient outcomes is evident through an inverse correlation between biomarker expression patterns in the s&i PTCs and PDAC patient survival rates. Key findings include upregulated MMPs and key ECM proteins (such as collagen 11 and TGFβ) under nutrient-avid conditions, known to be regulated by CAFs, alongside the concomitant reduction in E-cadherin expression associated with a poorly differentiated PDAC state under nutrient deprivation. Furthermore, elevated levels of hyaluronic acid (HA) and integrins in response to nutrient deprivation underscore the model's fidelity to the PDAC microenvironment. We also observed increased IL-6 and reduced α-SMA expression under poor nutritional conditions, suggesting a transition of CAFs from myofibroblastic to inflammatory phenotypes under a nutrient stress akin to in vivo niches. In conclusion, the s&i PTC represents a significant advancement in engineering clinically relevant 3D models of PDAC masses. It offers a promising platform for elucidating tumor-stroma interactions and guiding future therapeutic strategies to improve patient outcomes.

Ovarian Cancer Cell-Conditioning Medium Induces Cancer-Associated Fibroblast Phenoconversion through Glucose-Dependent Inhibition of Autophagy.

One aspect of ovarian tumorigenesis which is still poorly understood is the tumor-stroma interaction, which plays a major role in chemoresistance and tumor progression. Cancer-associated fibroblasts (CAFs), the most abundant stromal cell type in the tumor microenvironment, influence tumor growth, metabolism, metastasis, and response to therapy, making them attractive targets for anti-cancer treatment. Unraveling the mechanisms involved in CAFs activation and maintenance is therefore crucial for the improvement of therapy efficacy. Here, we report that CAFs phenoconversion relies on the glucose-dependent inhibition of autophagy. We show that ovarian cancer cell-conditioning medium induces a metabolic reprogramming towards the CAF-phenotype that requires the autophagy-dependent glycolytic shift. In fact, 2-deoxy-D-glucose (2DG) strongly hampers such phenoconversion and, most importantly, induces the phenoreversion of CAFs into quiescent fibroblasts. Moreover, pharmacological inhibition (by proline) or autophagy gene knockdown (by siBECN1 or siATG7) promotes, while autophagy induction (by either 2DG or rapamycin) counteracts, the metabolic rewiring induced by the ovarian cancer cell secretome. Notably, the nutraceutical resveratrol (RV), known to inhibit glucose metabolism and to induce autophagy, promotes the phenoreversion of CAFs into normal fibroblasts even in the presence of ovarian cancer cell-conditioning medium. Overall, our data support the view of testing autophagy inducers for targeting the tumor-promoting stroma as an adjuvant strategy to improve therapy success rates, especially for tumors with a highly desmoplastic stroma, like ovarian cancer.

Abstract PO4-28-03: Investigating the role of extracellular vesicles in tumor-stromal cell interactions in early-stage breast cancer invasion

Abstract Ductal carcinoma in situ (DCIS) is the most common form of early-stage breast cancer. Anywhere from 10%–30% of DCIS cases progress to invasive ductal carcinoma (IDC), resulting in poor prognosis and clinical challenges. Still, the cellular basis of this DCIS transition remains poorly understood. To escape the primary site during local invasion, DCIS cells navigate through the extracellular matrix and a diverse collection of stromal cells in the surrounding microenvironment. Cancer associated fibroblasts (CAFs) are a stromal cell population that can facilitate tumor invasion in part through extracellular matrix remodeling. CAFs at the primary tumor are largely derived from fibroblasts that have been reprogrammed to an activated state. In addition, several physical, cellular and metabolic changes within the tumor microenvironment (TME) are known to alter the breast cancer invasive capacity. Here we report that forced inhibition of cell invasion, by blocking the activity of known regulators of cell invasion in the ductal epithelial cell line, MCF10ADCIS.com, (herein referred to as DCIS.com), is accompanied by a marked reduction in extracellular vesicle (EV) secretion. Moreover, we show that DCIS.com cells conditioned with secreted EVs exhibit significantly enhanced invasive capacity relative to naïve DCIS.com cells. It is now well documented that breast and other tumor cells release heterogenous populations of EVs that can function as mediators of intercellular communication in the tumor microenvironment. EVs contain a host of bioactive cargo, such as membrane and cytosolic proteins, various RNA species including microRNAs, as well as dsDNA. These shed vesicles may deposit paracrine information and can also be taken up by stromal cells such as fibroblasts causing the recipient cells to undergo phenotypic changes that profoundly impact various facets of tumor progression. Conversely, EVs shed from stromal cells also affect tumor cell behavior. This unique form of intercellular crosstalk helps condition the TME and promotes both invasive and metastatic activity. In this regard, we found that incubation of human fibroblast cells with EVs shed from tumor cells leads to a loss of SMAD-7 expression (previously implicated in CAF reprogramming), and this loss is abrogated when shedding cells are depleted of miR-21 or regulators of miRNA trafficking to surface-derived EVs. Further, using spheroid invasion assays we demonstrate that fibroblast-derived EVs facilitate directed and leader-follower modes of migration, suggesting the possibility that CAFs likely deposit chemotactic cues to promote directional movement in DCIS. While the regulation of this behavior is still being explored, our observations suggest that EVs, in addition to facilitating distal proteolysis, may also support both autocrine and paracrine signaling during cell invasion. Finally, we are investigating how these pathways may also contribute to the ‘field effect’ of breast cancerization, a poorly understood process that introduces genetic and phenotypic changes in normal epithelia, potentially contributing to disease onset and/or recurrence. Citation Format: Madison Schmidtmann, Grace Richmond, Shireen Jayman, James Clancy, Crislyn D'Souza-Schorey. Investigating the role of extracellular vesicles in tumor-stromal cell interactions in early-stage breast cancer invasion [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-03.

Abstract PO4-28-10: Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model

Abstract Introduction: Our understanding of cancer has evolved over the last decades, with treatments increasingly targeting stromal cells in the tumor microenvironment (TME). The TME comprises transformed cancer cells and various non-cancerous cell types such as fibroblasts, macrophages, and endothelial cells. Despite this understanding, disrupting the tumor-stromal interactions are not targeted mainstream, as the stromal cells in the TME are genetically stable and can induce both beneficial and adverse effect on tumorigenesis. Thus, ex vivo tumor models that can faithfully recapitulate these critical tumor-stroma interactions are required to mechanistically understand the multifaceted reaction occurring within the TME. In this study, we established an innovative and multi-cellular TME on-a-chip model and assessed the synergistic effect of patient-derived Cancer Associated Fibroblast (CAF’s) and macrophages (M𝟇) on breast cancer progression. Materials and Methods: The influence of stromal components on cancer progression was assessed using the microfluidic model that allows spatial organization of various cell types. Tri-culture invasion model was established using Sum159 breast cancer cells, patient-derived CAFs, and THP-1 derived naïve (M𝟇) macrophages. Sum159 mixed with collagen and Matrigel forms the tumor region, while CAF’s and M𝟇 embedded in collagen form the stromal region. Functional and real-time assessments such as migration, morphometric analysis, and proliferation studies were conducted to understand the stromal influence on cancer cells. Gene expression analysis on an array of macrophage activation genes was performed to identify the polarized state of the macrophages after cellular interactions. Finally, Single Cell-RNA sequencing was carried out to unveil the drivers of stromal-immune crosstalk on invasion. Results and Discussion: Using the TME on-a-chip model, we compared cancer cell invasion across 4 different culture conditions namely monoculture (Sum only), co-culture (Sum+CAF, Sum+ M𝟇) and tri-culture (Sum+CAF+ M𝟇). Cancer cell migration was significantly increased in the triculture condition compared to others and stromal components also enhanced cancer cell proliferation. Hierarchical clustering of migration and morphological changes demonstrated that the co-culture conditions grouped together while CAF+ M𝟇 exerted the highest influence on cell migration and clustered separately. To understand the bi-directional communication between tumor and stroma, we assessed the polarized states of naïve macrophages using qPCR. Gene expression analysis showed that tumor-educated macrophages showed characteristics of M1 and M2 phenotypes and communicated reciprocally with tumor cells. Finally, to investigate the molecular mechanics of complex cellular interactions, we performed sc-RNA sequencing on cells extracted from the device and 2D cells (controls). Unsupervised clustering identified 11 different clusters, categorizing cells as Sum159 (tumor cells), CAFs (Stromal), and M𝟇 (Immune) based on cell-specific markers. Notably, we observed a noticeable shift in the clusters of Sum159 cells between 2D and 3D samples, emphasizing the difference between these two cultures. Conclusion: Using the TME on-a-chip, we confirmed the critical influence of microenvironmental components on tumor progression through functional assessment. Gene-expression analysis revealed bi-directional communication between noncancerous stromal components and tumor cells highlighting the importance of microenvironmental crosstalk. Furthermore, sc-RNA sequencing revealed the differences between 2D and 3D cultures. Ongoing work aims to perform transcriptomic and bioinformatic analysis on the sc-RNA sequencing data to gain a deeper understanding of the multifaceted communication within our platform. Citation Format: Kalpana Ravi, Lydia Sakala, Jin Park, Joshua LaBaer, Mehdi Nikkhah. Single-Cell Transcriptomic Assessment of Stromal and Immune Crosstalk on Breast Cancer Invasion Using 3D Tumor-On-Chip Model [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-10.

Microphysiological systems as models for immunologically 'cold' tumors.

The tumor microenvironment (TME) is a diverse milieu of cells including cancerous and non-cancerous cells such as fibroblasts, pericytes, endothelial cells and immune cells. The intricate cellular interactions within the TME hold a central role in shaping the dynamics of cancer progression, influencing pivotal aspects such as tumor initiation, growth, invasion, response to therapeutic interventions, and the emergence of drug resistance. In immunologically 'cold' tumors, the TME is marked by a scarcity of infiltrating immune cells, limited antigen presentation in the absence of potent immune-stimulating signals, and an abundance of immunosuppressive factors. While strategies targeting the TME as a therapeutic avenue in 'cold' tumors have emerged, there is a pressing need for novel approaches that faithfully replicate the complex cellular and non-cellular interactions in order to develop targeted therapies that can effectively stimulate immune responses and improve therapeutic outcomes in patients. Microfluidic devices offer distinct advantages over traditional in vitro 3D co-culture models and in vivo animal models, as they better recapitulate key characteristics of the TME and allow for precise, controlled insights into the dynamic interplay between various immune, stromal and cancerous cell types at any timepoint. This review aims to underscore the pivotal role of microfluidic systems in advancing our understanding of the TME and presents current microfluidic model systems that aim to dissect tumor-stromal, tumor-immune and immune-stromal cellular interactions in various 'cold' tumors. Understanding the intricacies of the TME in 'cold' tumors is crucial for devising effective targeted therapies to reinvigorate immune responses and overcome the challenges of current immunotherapy approaches.