Breast Cancer Patient-derived Xenograft Research Articles

Evaluation of Copanlisib in Combination with Eribulin in Triple-negative Breast Cancer Patient-derived Xenograft Models.

In this research, we demonstrated that the pan-PI3K inhibitor copanlisib enhanced the cytotoxicity of eribulin in a panel of TNBC PDX models. The improved tumor growth inhibition was irrespective of PI3K pathway alteration and was corroborated by the enhanced mitotic arrest and apoptotic induction observed in PDX tumors after combination therapy compared with each drug alone. These data provide the preclinical rationale for the clinical testing in TNBC.

PKMYT1 is a Marker of Treatment Response and a Therapeutic Target for CDK4/6 Inhibitor-Resistance in ER+ Breast Cancer.

Endocrine therapies (ET) with CDK4/6 inhibition are the standard treatment for estrogen receptor-α-positive (ER+) breast cancer, however drug resistance is common. In this study, proteogenomic analyses of 22 ER+ breast cancer patient-derived xenografts (PDXs) demonstrated that PKMYT1, a WEE1 homolog, is estradiol (E2) regulated in E2-dependent PDXs and constitutively expressed when growth is E2-independent. In clinical samples, high PKMYT1 mRNA levels associated with resistance to both ET and CDK4/6 inhibition. The PKMYT1 inhibitor lunresertib (RP-6306) with gemcitabine selectively and synergistically reduced the viability of ET and palbociclib-resistant ER+ breast cancer cells without functional p53. In vitro the combination increased DNA damage and apoptosis. In palbociclib-resistant, TP53 mutant PDX organoids and xenografts, RP-6306 with low-dose gemcitabine induced greater tumor volume reduction compared to treatment with either single agent. Our study demonstrates the clinical potential of RP-6306 in combination with gemcitabine for ET and CDK4/6 inhibitor resistant TP53 mutant ER+ breast cancer.

Abstract PO1-04-14: PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts

Abstract Paclitaxel is a first-line chemotherapy for hormone receptor positive (HR+) metastatic breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Response rate to paclitaxel ranges between 20-40%, and most patients progress due to intrinsic or acquired resistance, leaving them with limited treatment choices. Therapeutic strategies to overcome paclitaxel resistance and extend its clinical benefit are urgently needed. Paclitaxel-resistant tumors have increased expression of polo-like kinase 1 (PLK1), a serine-threonine-protein kinase that regulates mitosis and cell cycle progression. PLK1 has also been shown to mediate resistance to CDK4/6 inhibitor palbociclib in HR+ breast cancer. Onvansertib is an orally bioavailable, highly potent, and selective PLK1 inhibitor in clinical development. Onvansertib exhibited synergy in vitro and showed potent anti-tumor activity in vivo in combination with paclitaxel in ovarian cancer and triple negative breast cancer (TNBC) preclinical models. A phase 1b/2 clinical trial is ongoing to evaluate the safety and efficacy of onvansertib plus paclitaxel in advanced TNBC (NCT05383196). Here we investigated whether the potential of onvansertib and paclitaxel combination could be extended to HR+ breast cancer. Co-treatment with onvansertib and paclitaxel synergistically inhibited the viability of HR+ breast cancer cell lines (MCF-7, T-47D, EFM-19, CAMA-1, ZR-75-1). Compared to the single agents, the combination induced increased apoptosis. The combination was further tested in vivo in patient-derived xenograft (PDX) models. To this end, six HR+ breast cancer PDXs with intrinsic or acquired resistance to palbociclib were established from primary breast tumors (n=1) or metastatic bone biopsies (n=5). The PDX tumors were engrafted subcutaneously in nude mice and the effect of onvansertib (45 mg/kg, oral, 5 days a week) and paclitaxel (15-25mg/kg, IP, once a week) was investigated as monotherapy and in combination. The combination of onvansertib and paclitaxel was tolerated with minimal toxicity and showed enhanced anti-tumor activity compared to either agent alone in all 6 PDX models. Paclitaxel and onvansertib monotherapies had little to no anti-tumor activity in 4 out of 6 PDX models (HBCx124palboR25, HBCx-139 palboR5, HBCx-202 and HBCx-131). Importantly, the combination of onvansertib and paclitaxel induced strong anti-tumor activity in these 4 models. Tumor regression was observed in the 2 PDX models with acquired resistance to palbociclib, HBCx-124palboR25 and HBCx-139palboR5, with 62% (5/8 mice) and 54% (6/11 mice) complete response rates, respectively. The combination showed tumor stasis in HBCx-131 and induced tumor regression in HBCx-202, established from patient resistant to palbociclib. In 2 of the 6 PDXs, paclitaxel exhibited anti-tumor activity, inducing tumor regression in HBCx-137paboR26 and tumor stasis in HBCx-86. In comparison, the combination of onvansertib and paclitaxel had greater activity, inducing tumor regression in both models with a higher rate of complete response than the monotherapy. Complete response rates for paclitaxel versus the combination were 62% and 100% in the HBCx-137palboR26 model and 0% and 67% in the HBCx-86 model. Notably, the antitumor activity of the onvansertib and paclitaxel combination was very durable, showing a robust delay in tumor relapse after stopping therapy. Together, our data strongly support that combining paclitaxel with the PLK1 inhibitor onvansertib extends its benefit and overcomes paclitaxel resistance, and represents a promising therapeutic strategy for HR+ breast cancer patients after progression on a combination of endocrine and CDK4/6 inhibitor therapy. Citation Format: Maya Ridinger, Pierre Painsec, Sreeja Sreekumar, Dalia Gonzalez, Davis Klein, Laura Sourd, Elodie Montaudon, Paul Cottu, Tod Smeal, Elisabetta Marangoni. PLK1 Inhibitor Onvansertib Extends the Response and Overcomes Resistance to Paclitaxel in Palbociclib-resistant HR+ Breast Cancer Patient-derived Xenografts [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 PO1-04-14.

Abstract PO2-28-08: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract TNBC is one of the most aggressive subtypes of breast cancer. Combating chemotherapy resistance is critical to improving quality of care and reducing fatality among TNBC patients. In order to understand the mechanisms of chemoresistance in TNBC tumors, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy among tumors, and metastatic potential of certain tumors over others. Thus, this study utilizes spatially resolved transcriptomic (SRT) technology to profile the spatial interactions in the TNBC TME. With the goal of better understanding chemoresistance in TNBC and providing a proof-of-concept for new technology, we have launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Genomics Visium was performed on vehicle, AC-treated residual tumor (21 days post-Tx), and AC-treated regrown tumor (50 days post-Tx when tumors regrew to starting tumor volume). This study is divided into two parts: 1) development of a computational pipeline; 2) spatial colocalization analysis of residual and regrown tumors to understand chemoresistance. PART 1: Due to the lack of specialized tools for processing and sorting xenograft reads from SRT data, we developed the Xenomake pipeline, which combines a xenograft sorting algorithm (Xengsort) and spatial barcode demultiplexing pipeline to assign reads into the host and graft organisms for each spatial spot. RESULTS: Xenomake permits clustering the spatial spots into stroma-rich (enriched for mouse mRNAs), and epithelial-rich (enriched for human mRNAs) regions. We show that Xenomake can find differential cytokine production in the stroma and epithelium. Since PDX data separate the tumor into stroma and epithelium by organism, the pipeline enables fine-tuned downstream analysis such as stromal-stromal and stromal-epithelial interactions. Xenomake is thus generally applicable for SRT involving PDX samples. PART 2: Previously we detailed patterns of tumor regression into a residual tumor state, followed by uncontrolled regrowth in the absence of treatment in multiple PDX models of TNBC. Although using PDX models necessitates immune-compromised mice, several types of stromal populations are present and analyzable in these models. Thus, using organism-assigned reads processed by Xenomake, we computationally inferred the localization pattern of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial cells (ENDO), monocytes, and perivascular-like cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for the ~30 HALLMARK pathways across samples. We correlated the patterns of SPAM with stroma cell-type SLP to survey the stroma-epithelial interactions across samples. RESULTS: Stroma cells are generally distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and with CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Furthermore, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [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 PO2-28-08.

Abstract PO2-04-09: Combination of the PLK1 Inhibitor Onvansertib and the PI3Kα Inhibitor Alpelisib Overcomes Palbociclib Resistance in PIK3CA-mutated HR+ Breast Cancer

Abstract In the 1st line setting of hormone receptor-positive HER2 negative (HR+/HER2-) metastatic breast cancer, cyclin dependent kinase 4 and 6 (CDK4/6) inhibitors (palbociclib, abemaciclib and ribociclib) are typically used in combination with endocrine therapy. Though initially effective, acquired resistance to either CDK4/6 inhibitors or endocrine therapy eventually leads to disease relapse in most patients. Activating mutations in PIK3CA, the gene encoding alpha catalytic subunit of phosphatidylinositol-4,5-bisphosphat 3-kinase (PI3K) are found in approximately 40% of HR+ breast cancer patients and have been implicated in mediating resistance to CDK4/6 inhibitors. The PI3Kα inhibitor alpelisib is currently approved for PI3KCA-mutant HR+ breast cancer after progression on CDK4/6 inhibitors. However, drug-associated resistance mechanisms may limit its clinical benefit. Synergistic combination therapies have the potential to improve efficacy and overcome resistance mechanisms. Polo-like kinase 1 (PLK1) is a serine-threonine-protein kinase, key cell cycle regulator that controls G2/M entry and mitotic progression, and that has been shown to mediate resistance to palbociclib in HR+ breast cancer. Onvansertib is an oral and highly specific PLK1 inhibitor currently on clinical development in solid tumors and hematological malignancies. Here we evaluated the potential of onvansertib to increase the activity of alpelisib in PIK3CA-mutant HR+ breast cancer preclinical models. The combination of onvansertib and alpelisib synergistically inhibited cell viability and/or colony formation in three PI3KCA-mutant HR+ breast cancer cell lines, MCF-7 (PIK3CA E545K), EFM-19 (PIK3CA H1047L), and T-47D (PIK3CA 1047R). A significant increase in apoptosis was observed in cells treated with the combination compared to either agent alone. We next tested the combination of onvansertib and alpelisib in patient-derived xenograft (PDX) models. For this purpose, palbociclib-resistant PIK3CA-mutant HR+ breast cancer PDXs were established from primary breast tumor (PDX HBCx-86, PIK3CA E545K) or from metastatic bone biopsies of patients who had progressed on endocrine therapy plus palbociclib (HBCx-180, PIK3CA H1047R) or on PI3Kα inhibitor (HBCx-134palboR31, PIK3CA H1047R). PDX tumors were grafted subcutaneously in nude mice and mice were treated with vehicle, onvansertib (45 mg/kg, oral, 5 days a week), alpelisib (25mg/kg, oral, 5 days a week) or the combination. The combination was well tolerated and showed superior anti-tumor activity than the single agents in the 3 PDX models. In the HBCx-86 PDX, although none of the single agents showed activity, the combination induced potent tumor growth inhibition. In the metastasis-derived PDX HBCx-134palboR31, the combination treatment induced pronounced tumor regression in 62% of mice (5/8), with complete response in 25% (2/8), while mice treated with the single agents showed tumor progression. Finally, in the HBCx-180 PDX, established from a patient with primary resistance to palbociclib, the efficacy of the combination was greater and more durable, with significant survival advantage, compared to the monotherapies. Collectively, our preclinical findings suggest that co-targeting PLK1 and PI3Kα with onvansertib and alpelisib respectively, may constitute a promising therapeutic combination strategy for patients with PIK3CA-mutant HR+ breast cancer failing to respond to first-line standard of care therapies. Citation Format: Sreeja Sreekumar, Pierre Painsec, Davis Klein, Dalia Gonzalez, Laura Sourd, Elodie Montaudon, Tod Smeal, Elisabetta Marangoni, Maya Ridinger. Combination of the PLK1 Inhibitor Onvansertib and the PI3Kα Inhibitor Alpelisib Overcomes Palbociclib Resistance in PIK3CA-mutated HR+ Breast Cancer [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 PO2-04-09.

Abstract 1986: Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts

Abstract Sacituzumab Govitecan (SG) is an antibody drug conjugate that targets the epithelial glycoprotein Trop-2. SG has been approved for the treatment of patients with triple negative breast cancer (TNBC) or estrogen receptor positive (ER+) breast cancer after they have received at least two previous systemic treatments. The objectives of this study were to identify biomarkers of SG response using breast cancer patient-derived xenografts (PDXs) and determine if SG efficacy changes when cells become carboplatin resistant (CR). Analysis of transcriptomic and proteomic levels of Trop2 using bulk RNAseq, scRNA-seq, and tandem mass tag spectrometry found that overall ER+ PDXs had significantly lower RNA (p-value less than 0.001) and protein (p-value less than 0.001) expression of Trop-2 than TNBC PDXs. Immunohistochemical assessment of 42 TNBC and 27 ER+ patient tumor samples identified variation in Trop-2 expression; within each cohort, expression varied from highly positive to completely negative. To test the association of Trop-2 with drug responsiveness, cells from 19 different PDXs were cultured in vitro and administered increasing doses of SG. TNBC cells were more responsive to SG treatments compared to ER+ cells (p-value equaling 0.005). Correlation analysis identified a positive relationship of drug response with protein abundance which was stronger in TNBC samples. In vivo studies with 7 TNBC PDXs found that over 70% were highly susceptible to SG treatments, with some tumors being completely eradicated or exhibiting a total inhibition of tumor growth which resulted in a long-term durable response after 10 weeks of treatment. In vivo across all the PDXs, Trop-2 expression alone did not strongly correlate with SG treatment success. Interestingly, CR sublines derived from 2 carboplatin sensitive PDXs were significantly less responsive to SG than their parental PDXs (p-value less than 0.001). These two CR sublines were also significantly less sensitive to the majority of 555 other drugs identified by the NCI compared to their parental PDXs. Ongoing studies are defining mechanisms of reduced SG efficacy in CR models. Overall, these studies find that SG is highly effective in many TNBC PDX models and should be utilized earlier in treatment protocols, especially in metastatic patients. Citation Format: Carson J. Walker, Julia E. Altman, Emily K. Zboril, Rachel K. Myrick, Nicole S. Hairr, David C. Boyd, Bin Hu, Mikhail G. Dozmorov, J. Chuck Harrell. Acquisition of carboplatin resistance corresponds with reduced sacituzumab govitecan efficacy in triple negative breast cancer patient derived xenografts [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 1986.

Abstract 3876: A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy

Abstract Combating chemotherapy resistance in TNBC is critical to improving quality of care and reducing fatality. To understand chemoresistance, it has been proposed that the spatial interactions between cell types in the tumor microenvironment (TME) could offer insights into the differential response to therapy and metastatic potential. Thus, this study utilizes spatially resolved transcriptomics (SRT) to profile the spatial interactions in TNBC TME. With the goal of understanding chemoresistance and providing a proof-of-concept for new technology, we launched a longitudinal SRT study of a TNBC PDX of residual disease before, during, and after adriamycin and cyclophosphamide (AC) treatment (Tx). SRT by 10X Visium was performed on Veh, AC-residual tumor (21 d post-Tx), and AC-regrown tumor (50 d post-Tx when tumors regrew). This study is divided into two parts. 1. Due to the lack of specialized tools for processing xenograft reads from SRT, we developed the Xenomake pipeline, which combines xenograft sorting and spatial barcode demultiplexing to assign reads into the host and graft organisms per spatial spot. It permits clustering the spots into stroma-rich (mouse), and epithelial-rich (human) regions. We show that it can find differential cytokines in the stroma (S) and epithelium (E), and finding SS, EE, and SE interactions. 2. Although using PDX necessitates immune-compromised mice, several types of stromal populations are present and analyzable. Thus, using Xenomake, we inferred the localization of stromal cell types in Visium spots including cancer associated fibroblasts (CAF), macrophages (MP), endothelial (ENDO), monocytes, and perivascular cells. We compared the spatial localization profiles (SLP) of stromal cell types across samples. With human reads, we computed the spatial pathway activity map (SPAM) for HALLMARK pathways. We correlated SPAM with stroma cell type SLP to survey stroma-epithelial interactions. Stroma cells are distributed in the tumor periphery in vehicle and AC50, while in AC21 there is a notable increase in stroma abundance and stroma infiltration within tumor mass. SLP of MP (Cd68+ and Csf1r+) is correlated with ENDO (Pecam1+), and CAF (Acta2+ and Pdgfrb+). Additionally, all 3 cell types are correlated with Vim and Cd44 expression. Although all samples show enrichment of MP, ENDO, and CAF, they interact differently with pathway activities of adjacent epithelial cells. In vehicle and AC50, the 3 cell types are colocalized with OXPHOS, MYC targets, E2F pathway, while in AC21, a switch to colocalization with EMT is observed. Further, hypoxia response and glycolysis display anti-correlation with stroma cell types, meaning that these pathway activities are further away from stroma and occupy distinct territories. The results suggest stroma-tumor metabolic crosstalk and ways of targeting residual disease. Citation Format: Benjamin Strope, Katherine Pendleton, William Bowie, Gloria Echeverria, Qian Zhu. A spatial transcriptomic study of a triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model of residual disease refractory to conventional chemotherapy [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 3876.

Abstract 1750: Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration

Abstract Breast cancer's complex transcriptional landscape requires a deep understanding of sample and cell diversity to identify effective treatments. In this study, we amalgamate single-cell RNA sequencing data from breast cancer patient-derived xenografts (PDX), organoids, cell lines, patient tumors and reduction mammoplasties resulting in a comprehensive dataset of 117 samples with 506,719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 enriched (HER2E), and triple-negative breast cancer (TNBC) subtypes. We aimed to delineate similarities and distinctions across model systems and patient samples while also exploring stratification of therapeutic drug efficacy based on subtype proportions within tumors. Mammary tumor PDXs, organoids, and established cell lines exhibited higher proliferation and lower heterogeneity observed via UMAP dimensionality reduction compared to most patient tumors or normal breast epithelium. TNBCs had elevated proliferative and pro-metastatic signatures compared to HR+ and HER2E samples. Interestingly, compared to matched PDX tumors, organoids from these same models were found to exhibit stark differences in gene expression, including upregulation of metabolically active aldo-keto reductase family genes, highlighting differences in the model systems with implications for pre-clinical drug testing. Single-cell tumor subtyping analyses with scSubtype and TNBCtype methods found that therapeutically treated samples had shifts in the proportions of cell-wise subtype annotations when compared to matched untreated samples. Similarly, patient lymph node metastasis when compared with matched primary tumors were significantly linked to decreases in Basal-like and HER2-enriched cell-wise annotations in untreated ER+ samples. In vitro assessment of anti-cancer compounds on PDX cells showed significant correlation of subtype proportion with cell viability following treatment with targeted therapeutic agents. This subtyping methodology offers a powerful tool to monitor the evolving gene expression landscape within samples and predict responses to therapeutic agents. We present here a dynamic approach to cell-wise sample annotation and a substantial multi-model dataset for use facilitating informed decision-making in preclinical research and therapeutic development. Citation Format: Julia E. Altman, Carson J. Walker, Emily K. Zboril, Nicole S. Hairr, Rachel K. Myrick, David C. Boyd, Jennifer E. Koblenski, Madhavi Puchalapalli, Bin Hu, Mikhail G. Dozmorov, Xi Chen, Yunshun Chen, Charles M. Perou, Brian D. Lehmann, Jane E. Visvader, Amy L. Olex, J. Chuck Harrell. Decoding breast cancer: Unraveling subtype and model differences through multi-model single-cell RNA sequencing data integration [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 1750.

Abstract 907: ScreenDL: A transfer learning framework integrating tumor omics and functional drug screening for personalized clinical drug response prediction

Abstract Precision oncology hinges on accurate prediction of patient-specific treatment response from tumor molecular inputs. While deep learning (DL) models achieve state-of-the-art response prediction in cell lines, existing methods do not readily translate to the clinic where training data is limited. Here, we have developed and validated ScreenDL, a novel DL framework designed explicitly for use in clinical precision oncology applications. The underlying architecture of ScreenDL consists of two fully connected branches dedicated to learning rich embeddings of a drug’s chemical structure and a tumor’s transcriptomic profile respectively. These tumor omic and drug molecular embeddings are fed into a shared response prediction subnetwork which outputs predicted ln(IC50) values. The ScreenDL training schema incorporates three phases, each designed to improve clinical response prediction: (1) an initial pretraining phase leverages large-scale cell line pharmacogenomic datasets to extract patterns/relationships linking tumor omics and drug response; (2) subsequent transfer learning integrates pharmacogenomic data from patient-derived models of cancer, adapting the pretrained model to a more patient-relevant context; (3) patient-level omics and drug screening data (e.g., from matched patient-derived organoid (PDO) models) is integrated through patient-specific fine-tuning, generating a patient-specific response prediction model. Importantly, biomaterial from surgical tumor resection is often available for organoid establishment and patient-specific drug screening in practical clinical scenarios. To assess the utility of our ScreenDL framework for clinical response prediction, we applied ScreenDL to predict treatment response in 50 advanced/metastatic triple negative breast cancer (TNBC) patient-derived xenograft models (PDX). We leveraged tumor omics profiles for each PDX and drug screening data from matched PDX-derived organoids (PDxOs), mirroring the combination of patient-level tumor omic characterization and functional drug screening in matched PDOs, a protocol currently being piloted in Functional Precision Oncology trials at the University of Utah Huntsman Cancer Institute. After cell line pretraining, ScreenDL achieves a modest median Pearson correlation per drug of 0.15 relative to 0.03 for the leading compared model. However, transfer learning and subsequent PDX-specific fine-tuning significantly improve performance, producing median Pearson correlations per drug of 0.39 and 0.51, respectively. These results demonstrate the ability of our ScreenDL framework to dramatically improve response prediction in clinically relevant domains, bringing DL-based precision treatment selection closer to clinical application. Citation Format: Casey Sederman, Tonya Di Sera, Yi Qiao, Xiaomeng Huang, Bryan E. Welm, Alana L. Welm, Gabor Marth. ScreenDL: A transfer learning framework integrating tumor omics and functional drug screening for personalized clinical drug response prediction [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 907.

Abstract 6429: Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer

Abstract Objective: In the cancer research, a highly promising in vivo platform for personalized treatment involves the adoption of an advanced approach—specifically, the grafting of patient-derived xenografts (PDX) onto humanized mouse models. These models capture distinctive aspects of interaction with the human immune system and the tumor microenvironment (TME). In this study, we have engineered humanized triple-negative breast cancer (TNBC) PDX models to observe alterations in differentially expressed genes, thereby enhancing our understanding of the intricacies of personalized treatment in this context. Method: We utilized two TNBC PDX models established for the humanized models, where CD34+ hematopoietic stem cells were transplanted into immunodeficient mice. RNA sequencing was performed to identify altered gene expression in humanized PDX models compared to NSG PDXs. Additionally, we generated stable cell lines overexpressing TMEM176B in MDAMB231 and Hs578T cells, and we knocked down TMEM176B in 4T1 cells using recombinant techniques Results: In the humanized model, the growth of PDX tumors did not show a significantly faster or markedly different rate compared to previous studies. However, upon conducting RNA sequencing in the PDX model, the expression of the TMEM176B gene was consistently downregulated. Consequently, we transfected and observed that the increased expression of TMEM176B in MDAMB231 and Hs578T cell lines resulted in enhanced cell proliferation in vitro. Transplanting MDAMB231 cells overexpressing TMEM176B into athymic nude mice resulted in accelerated tumor growth. The study further investigates the expression of TMEM176B in an immune-competent environment by knocking down the gene in 4T1 cells or transplanting the control group into a balb/c mouse model. Tumors with TMEM176B knockdown exhibited inhibited growth, increased CD3+ T cell infiltration, and significant inhibition when treated with anti-PD-L1 compared to the control group. Conclusion: This research demonstrates the utility of PDX models, particularly humanized PDX models, in studying the complexities of cancer biology and the tumor microenvironment. The findings regarding TMEM176B and its role in tumorigenesis and immune responses in triple-negative breast cancer provide valuable insights for further research and potential therapeutic interventions. Citation Format: Yujeong Her, Jihui Yun, Woohang Heo, Jong-Il Kim, Han-Byoel Lee, Wonshik Han, Hyeong-Gon Moon. Critical role of elevated TMEM176B gene expression in humanized patient-derived xenografts mouse models of triple negative breast cancer [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 6429.

Abstract 2826: Examples of translational preclinical models to mimic cancer drug resistance

Abstract Drug resistance is becoming a major obstacle when treating patients with cancer. Cancer is characterized by an uncontrolled growth of abnormal cells with permanent genome alterations, which lead to severe mutations including resistance mechanisms. Mutations can occur in different resistance pathways, such as inhibition of drug transport, DNA damage repair, drug efflux, EMT (epithelial-mesenchymal transition), drug target alteration and cell death inhibition. Resistance mechanisms are now well studied in vitro, based on mutated cell lines for specific resistance pathways. There is however a gap in our knowledge of resistance mechanisms observed in patient tumor cells. We are challenged to discover cancer resistance treatments in patients and need predictive models of clinical situations. Animal tumor models are the best translational tools to study cancer drug resistance, especially when they closely mimic human pathologies. Such models can bridge the gap between our understanding of cancer resistance in vitro and in tumor patients. In this poster we show different pre-clinical experiments, simulating clinical translational approaches, that led to novel and unique tumor resistant models. Patient Derived Xenograft (PDX) models: Using our breast cancer PDX collection we showed that after repeated treatment with a spindle poison, tumor-bearing mice started to develop resistance and continued tumor growth under drug pressure. Following this successful approach, we initiated the development of a novel ER2-expressing PDX breast tumor model, resistant to hormone therapy by repeated Fulvestrant treatment. Using our colon cancer PDX collection we were able to recapitulate clinical situations by correlating PDX molecular profiles and drug sensitivity. We confirmed the key role of a KRAS mutation in cetuximab resistance. Syngeneic model: In this model, animals were dosed with anti-PDL1 as first line of treatment. All animals were then defined as initial responders or non-responders at the end of the dosing schedule. Following this, a new treatment protocol was initiated with a Tyrosine Kinase inhibitor to study the effect of second line drug treatment in responder and non-responder arms. Human model: In this experiment, we performed a first line Avastin treatment on human colon carcinoma tumor-bearing mice. A tumor resistance to Avastin was observed. During the second line of treatment with a tyrosine kinase inhibitor, we observed an antitumor activity on Avastin resistant animals. In each of these case studies, we developed a novel tumor-resistant mechanism model. In some tumor models, we showed the added value of treating animals with new drugs as second line treatment, after establishing resistance to the first line standard of care treatment. Citation Format: Marc Hillairet de Boisferon, Nicolas Hoffmann, Elodie Rajon, Ismahène Benzaid. Examples of translational preclinical models to mimic cancer drug resistance [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 2826.

Abstract 5911: In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer

Abstract Introduction: Antibody and peptide-conjugated drugs are rapidly entering the clinic for the treatment of patients with early and advanced breast cancer. These drugs are designed to achieve more targeted delivery of cytotoxic agents to tumor cells with robust clinical activity. ARB-1-6, a sortilin (SORT1) targeted peptide conjugated to the cytotoxic agent MMAE, has demonstrated pre-clinical activity and tolerability in cell line xenografts of breast cancer. To further characterize its spectrum of antitumor activity and biomarker correlates we evaluated ARB-1-6 in a collection of clinically and genomically annotated breast cancer patient-derived xenografts (PDX). Methods: Breast cancer PDX models were generated and propagated under IRB approved protocols [14-8358]. ARB-1-6 was dosed in tumor-bearing SCID mice by tail vein injection at 3 mg/kg weekly x 4 doses. Tumor volume was measured twice weekly until day 35 or until models reached humane endpoints, after which tumors were collected. Animals were weighed regularly and assessed for treatment-related toxicity. Results: 7 models (5 TN and 2 HR) have completed efficacy assessment to date. 3/7 models were derived from treatment naïve patients while 4/7 were derived from pre-treated patients (Table 1). Complete response (mRECIST) was seen in 5/7 (71.4%) models with stable disease occurring in 2/7 (28.6%). ARB-1-6 was well tolerated with no weight loss observed. Complete tumor regression was seen in a PDX derived from a patient with clinical resistance to a TROP2 antibody drug conjugate (ADC). Antitumor activity was observed in models with low SORT1 RNA expression; SORT1 IHC and other correlatives studies are underway. Conclusion: ARB-1-6 demonstrates substantial preclinical activity in PDX models at doses that are well tolerated in mice. Treatment of additional models is ongoing. Full results and correlative analyses including relationships between antitumor activity and target protein expression will be presented. PDX Model Characteristics and Patient Treatment PDX Subtype Pt. Treatment 1 TN AC-T 2 TN FEC-D, Gem+Carbo 3 TN AC-T 4 TN No 5 TN No 6 HR No 7 HR TC, ET, Cape, Gem+Cis, TROP2-ADC Citation Format: Mitchell J. Elliott, Meghan Mcguire, Jennifer Silvester, Chantal Tobin, Samah El Ghamrasni, Francine Lui, Andrew Zhai, Ozge Yoluk, Aron Broom, Tracy Stone, Glenn Butterfoss, Serban Popa, Tianyu Lu, Chris Ing, David White, David W. Cescon. In vivo efficacy of a novel peptide-conjugated drug in patient-derived xenograft models of breast cancer [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 5911.

Abstract 742: PYX-201, a stroma-targeting ADC composed of an anti-EDB+FN antibody conjugated to Auristatin0101, demonstrates strong anti-tumor efficacy across multiple human cancer indications in pre-clinical PDX tumor models

Abstract Stroma is crucial to support solid tumor growth, metastasis and resistance to treatment. While most antibody-drug conjugates (ADCs) directly bind to cancer cells, PYX-201, an investigational drug, is designed to target tumor stroma by binding to the extra-domain B splice variant of fibronectin (EDB+FN), a matrix protein abundantly expressed in the tumor microenvironment of many solid tumors with absent or low expression in normal adult tissues. In vitro studies demonstrated PYX-201 was strongly cytotoxic to EDB+FN positive Caki2 cells, but not to EDB+FN negative HT29 cells. In addition, PYX-201 was 30-times more potent than a non-targeted ADC control, demonstrating PYX-201 cytotoxic effects were EDB+FN dependent. Furthermore, when Caki2 and HT29-luciferase cancer cells were co-cultured, the luciferase activity was used to quantify the amount of EDB+FN negative HT29 cells only. Interestingly, PYX-201 was able to kill EDB+FN negative cancer cells in a co-culture assay as shown by a decrease in luciferase activity, demonstrating PYX-201 bystander activity. Pre-clinical mouse model studies were conducted to evaluate PYX-201 anti-tumor efficacy and pharmacokinetic (PK) properties. First, a comprehensive patient-derived xenograft (PDX) mouse model trial was designed to evaluate the anti-tumor efficacy of PYX-201. To fully represent the diversity and characteristics of human tumors, PDX models were pre-selected from ten solid tumor indications based on different levels of EDB+FN protein expression and stromal density. PYX-201 (Q4D × 4 at 3mg/kg) was well tolerated, and strong anti-tumor responses (% Tumor Growth Inhibition [TGI] >90%) were observed in PDX models across various cancer indications. Potential correlations between EDB+FN protein expression with anti-tumor activity will be evaluated. Remarkably, long-term anti-tumor efficacy of PYX-201 was observed as tumors did not relapse even after >100 days in a breast cancer PDX model. Additionally, plasma samples were analyzed from a cell line derived xenograft mouse model after a single dose of PYX-201 to determine its PK profile. At 3 mg/kg, the maximum concentration (Cmax) was 28.1 μg/mL, area under the curve (AUC) was 1,732.7 h*μg/mL, with a low clearance of 1.5 mL/h/kg, and the half-life of the mAb from PYX-201 was calculated at 81.2 h. These PK parameters were associated with strong PYX-201 anti-tumor efficacy (% TGI >90%) in the H1975 xenograft model. Altogether, PYX-201 was well tolerated and demonstrated strong anti-tumor efficacy in a variety of pre-clinical human PDX models. PYX-201 is a promising and innovative ADC which is currently under investigation in a Phase I clinical trial (NCT05720117). Citation Format: Nicolas Severe, Amanda Facklam, Liz McMichael, Biplab Das, Sara Lewandowski, Justin Trickett, Liyang Diao, Chengfeng Merriman, Chuan Shen, Jianwen Feng, Shawn Harriman, Marsha Crochiere, Philipp Steiner, Jan Pinkas. PYX-201, a stroma-targeting ADC composed of an anti-EDB+FN antibody conjugated to Auristatin0101, demonstrates strong anti-tumor efficacy across multiple human cancer indications in pre-clinical PDX tumor models [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 742.

PROM2 overexpression induces metastatic potential through epithelial-to-mesenchymal transition and ferroptosis resistance in human cancers.

Despite considerable therapeutic advances in the last 20 years, metastatic cancers remain a major cause of death. We previously identified prominin-2 (PROM2) as a biomarker predictive of distant metastases and decreased survival, thus providing a promising bio-target. In this translational study, we set out to decipher the biological roles of PROM2 during the metastatic process and resistance to cell death, in particular for metastatic melanoma. Methods and results: We demonstrated that PROM2 overexpression was closely linked to an increased metastatic potential through the increase of epithelial-to-mesenchymal transition (EMT) marker expression and ferroptosis resistance. This was also found in renal cell carcinoma and triple negative breast cancer patient-derived xenograft models. Using an oligonucleotide anti-sense anti-PROM2, we efficaciously decreased PROM2 expression and prevented metastases in melanoma xenografts. We also demonstrated that PROM2 was implicated in an aggravation loop, contributing to increase the metastatic burden both in murine metastatic models and in patients with metastatic melanoma. The metastatic burden is closely linked to PROM2 expression through the expression of EMT markers and ferroptosis cell death resistance in a deterioration loop. Our results open the way for further studies using PROM2 as a bio-target in resort situations in human metastatic melanoma and also in other cancer types.

The Tumor Growth Inhibitory Effect of a Standardized Extract of Cultured Lentinula edodes Mycelia Using Patient Derived Xenograft Model.

Patient derived xenograft (PDX) is a powerful tool to confirm pharmacological efficacy in non-clinical studies for the development of various drugs including anti-cancer agents and therapeutic research. A standardized extract of cultured Lentinula edodes mycelia, a product name AHCC® is produced by Amino Up Co., Ltd. (Sapporo, Japan). In this study, we investigated the inhibitory effect of AHCC® on the growth of tumor PDX in Super SCID (severe combined immunodeficiency) mice. Effects of AHCC® and BCG administration on the growth of renal cancer PDX implanted in Super SCID mice were evaluated by PDX growth curve. Tendency for the effects on the growth of renal cancer PDX in Super SCID by administration of AHCC® and BCG before implanting the PDX were demonstrated. The effects of the oral administration of AHCC® on the growth of renal, invasive and non-invasive breast cancer PDX in Super SCID mice were studied. In Super SCID mice transplanted with renal cancer PDX, AHCC® significantly suppressed tumor proliferation from the day 48 to 83 after transplantation. In two types of breast cancer PDX, tendency of the growth inhibitory effects of AHCC® were shown by PDX growth curve. Significant inhibitory effect was found at only one time point for during proliferation in each PDX. Super SCID-PDX model has the potential to be a useful tool to investigate for the effect of functional foods.

Toxoflavin analog D43 exerts antiproliferative effects on breast cancer by inducing ROS-mediated apoptosis and DNA damage

Triple-negative breast cancer (TNBC) is regarded as the deadliest subtype of breast cancer because of its high heterogeneity, aggressiveness, and limited treatment options. Toxoflavin has been reported to possess antitumor activity. In this study, a series of toxoflavin analogs were synthesized, among which D43 displayed a significant dose-dependent inhibitory effect on the proliferation of TNBC cells (MDA-MB-231 and HCC1806). Additionally, D43 inhibited DNA synthesis in TNBC cells, leading to cell cycle arrest at the G2/M phase. Furthermore, D43 consistently promoted intracellular ROS generation, induced DNA damage, and resulted in apoptosis in TNBC cells. These effects could be reversed by N-acetylcysteine. Moreover, D43 significantly inhibited the growth of breast cancer patient-derived organoids and xenografts with a favorable biosafety profile. In conclusion, D43 is a potent anticancer agent that elicits significant antiproliferation, oxidative stress, apoptosis, and DNA damage effects in TNBC cells, and D43 holds promise as a potential candidate for the treatment of TNBC.

DSBSO-Based XL-MS Analysis of Breast Cancer PDX Tissues to Delineate Protein Interaction Network in Clinical Samples.

Protein-protein interactions (PPIs) are fundamental to understanding biological systems as protein complexes are the active molecular modules critical for carrying out cellular functions. Dysfunctional PPIs have been associated with various diseases including cancer. Systems-wide PPI analysis not only sheds light on pathological mechanisms, but also represents a paradigm in identifying potential therapeutic targets. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for defining endogenous PPIs of cellular networks. While proteome-wide studies have been performed in cell lysates, intact cells and tissues, applications of XL-MS in clinical samples have not been reported. In this study, we adopted a DSBSO-based in vivo XL-MS platform to map interaction landscapes from two breast cancer patient-derived xenograft (PDX) models. As a result, we have generated a PDX interaction network comprising 2,557 human proteins and identified interactions unique to breast cancer subtypes. Interestingly, most of the observed differences in PPIs correlated well with protein abundance changes determined by TMT-based proteome quantitation. Collectively, this work has demonstrated the feasibility of XL-MS analysis in clinical samples, and established an analytical workflow for tissue cross-linking that can be generalized for mapping PPIs from patient samples in the future to dissect disease-relevant cellular networks.

Abstract B052: The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer

Abstract Introduction: Previously we discovered that a novel metastasis-initiating cell (MIC) population is marked by LMO2, a transcriptional adaptor protein in hematopoietic stem cells, and a T-cell oncogene. Knocking down LMO2 did not affect the growth of primary tumors, but did significantly reduce the number of circulating tumor cells and lung metastases. Transcriptional analysis of LMO2 knockdown shows the HALLMARK_DNA_REPAIR pathway is upregulated, suggesting that LMO2 is involved in regulating DNA repair in breast cancer cells. Interestingly, a previous study reported that in diffuse large B-cell lymphoma high LMO2 expression results in defective repair of double-stranded DNA breaks (Parvin et al., Cancer Cell, 36, 3 [2019]). We hypothesize that in breast cancer LMO2 expression is regulating the choice between homology-directed repair (HDR), an error-free method of DNA repair, and non-homologous end-joining (NHEJ), a more error-prone method. Cells that use NHEJ to repair DNA are dependent on poly (ADP-ribose) polymerase (PARP) and can be targeted with PARP inhibitors. LMO2 could thus be used as a new biomarker for benefit from PARP inhibition. Methods: LMO2 was identified using single-cell RNAseq data from human breast cancer patients. In vitro and in vivo assays using breast cancer cell lines and patient-derived xenografts were used to characterize the effect of LMO2 on metastasis. To study the role of LMO2 in the DNA damage response, we used MDA-MB-468 and HCC1806 cells with control and 2 independent shRNAs targeting LMO2. Immunofluorescent staining for γH2AX, 53BP1, and RAD51 was performed to measure changes in DNA damage and repair markers. Control and LMO2 knockdown cells were treated with olaparib, with cellular proliferation measured with a WST-1 assay and colony formation measured with crystal violet staining. Results and Conclusion: Knocking down LMO2 expression results in decreased NHEJ and increased HDR in MDA-MB-468 and HCC1806 cells. There is no significant difference in DNA damage overall as measured by γH2AX foci/nucleus. LMO2 knockdown in MDA-MB-468 cells reduces sensitivity to PARP inhibition with olaparib, as measured by an increase in the IC50 values. Our preliminary data suggest that LMO2 expression likely regulates the choice between NHEJ and HDR. LMO2 expression introduces a bias towards NHEJ, which sensitizes cells to PARP inhibition. This suggests that LMO2+ cells could be targeted with PARP inhibition, which could thus be used to target metastasis-initiating cells. Citation Format: Isobel Fetter, Veronica Haro-Acosta, Shaheen Sikandar. The role of LMO2 in DNA damage repair pathway choice in metastatic breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B052.

Abstract B067: Identification of Gemcitabine-resistant Populations using scRNA-Sequencing in Triple Negative Breast Cancer Patient-Derived Xenografts

Abstract Despite major advances in the treatment of breast cancer (BC), it remains the most diagnosed and second deadliest cancer among American women. BC consists of distinct clinical subtypes, including estrogen receptor or HER2 receptor-positive and triple-negative breast cancer (TNBC) that lacks these receptors preventing the use of established precision therapies and the standard of care remains neoadjuvant chemotherapy (NACT), followed by surgery, radiation and emerging combinations with immunotherapy. While NACT is effective in some patients, up to 50% of patients are intrinsically unresponsive or acquire resistance. These patients demonstrate the highest rates of recurrence and distant metastasis. It is known that chemotherapies can effectively eliminate proliferating tumor cells, but a small population of drug-tolerant persister (DTPs) cells can remain and are thought to play a key role in relapse and resistance. We hypothesized that chemotherapy influences TNBC tumor plasticity by exerting selective pressures leading to the outgrowth of resistant subpopulations with the greatest survival advantage. To evaluate this hypothesis, we generated in vivo models of disease progression and chemotherapy resistance and investigated the plasticity of tumor cell subpopulations by single-cell RNA sequencing. We developed PDX models from a multi-drug resistant primary TNBC treated with standard-of-care chemotherapy and its patient-matched lung metastasis. To model response and resistance at the metastatic stage, we challenged this metastasis PDX with several cycles of Gemcitabine. We obtained residual disease that relapsed and eventually developed resistance, mirroring the patient response. We performed single-cell RNA sequencing (scRNAseq) of these models using a droplet-based technology from 10X Genomics. This scRNAseq data was used to compare the changes in the proportions of cellular subpopulations in each model. Our data show that the rebound model presents greater similarity to the untreated metastasis, while the resistant model significantly differs in cell population expression profiles. Interestingly, the residual disease demonstrates an intermediated state with similarities to both the untreated metastasis and the resistant model. We identified populations with hypoxic signatures in the primary tumor, its matched metastasis and the residual, rebound and resistant models. Immunohistochemistry and Nanostring GeoMx Digital Spatial Profiler validated these populations in these models. In addition, we have identified other varying populations and are currently investigating their cellular mechanisms and gene expression patterns. Using scRNA-sequencing to understand the clonal expansion of resistant subpopulations following chemotherapy reveals distinctive resistant cell features enabling the identification of the vulnerabilities of these tumors. Citation Format: Sandrine Busque, Constanza Martinez Ramirez, Ariel Madrigal Aguirre, Paul Savage, Anie Monast, Anne-Marie Fortier, Gerardo Zapata, Atilla Omeroglu, Jamil Asselah, Nathaniel Bouganim, Sarkis Meterissian, Francine Tremblay, Ari Meguerditchian, Yasser Riaz Alhosseini, Hamed S. Najafabadi, Hellen Kuasne, Morag Park. Identification of Gemcitabine-resistant Populations using scRNA-Sequencing in Triple Negative Breast Cancer Patient-Derived Xenografts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B067.

Adoptive cell transfer therapy with ex vivo primed peripheral lymphocytes in combination with anti-PDL1 therapy effectively inhibits triple-negative breast cancer growth and metastasis

BackgroundAdoptive cell transfer cancer immunotherapy holds promise for treating disseminated disease, yet generating sufficient numbers of lymphocytes with anti-cancer activity against diverse specificities remains a major challenge. We recently developed a novel procedure (ALECSAT) for selecting, expanding and maturating polyclonal lymphocytes from peripheral blood with the capacity to target malignant cells.MethodsImmunodeficient mice were challenged with triple-negative breast cancer cell lines or patient-derived xenografts (PDX) and treated with allogeneic or autologous ALECSAT cells with and without anti-PDL1 therapy to assess the capacity of ALECSAT cells to inhibit primary tumor growth and metastasis.ResultsALECSAT mono therapy inhibited metastasis, but did not inhibit primary tumor growth or prolong survival of tumor-bearing mice. In contrast, combined ALECSAT and anti-PDL1 therapy significantly inhibited primary tumor growth, nearly completely blocked metastasis, and prolonged survival of tumor-bearing mice.ConclusionsCombined ALECSAT and anti-PDL1 therapy results in favorable anti-cancer responses in both cell line-derived xenograft and autologous PDX models of advanced triple-negative breast cancer.