Small cell lung cancer (SCLC) typically displays a “cold” tumor microenvironment with a paucity of immune infiltrate. Neuroendocrine SCLC cells also profoundly repress MHC-I expression, rendering them vulnerable to NK cell-mediated cytotoxicity. Here, we confirm that neuroendocrine SCLC cells are sensitive to NK cell-mediated attack, yet the quantitative spatial profiling of the SCLC immune microenvironment in patient samples reveals that effector immune cells, including NK cells, are excluded from MHC-Ilow/neg SCLC regions. To study this biology, we develop dynamic single-cell RNA sequencing of microphysiological immune tumor environments (DynaMITE-seq) and integrate findings with spatial transcriptomics in patient tissue, unveiling the microvasculature as a major checkpoint restricting NK cell extravasation/recruitment. We demonstrate that the activation of vascular Stimulator of Interferon Genes (STING) signaling restores NK cell infiltration and killing of neuroendocrine SCLC, suggesting a strategy to overcome this key SCLC immunologic barrier and prime therapeutic response to DLL3-targeted CAR-NK cell therapy.

cGAS-STING signaling can promote antitumor immunity, and tumor cell STING is suppressed in a variety of cancer subtypes that resist immune checkpoint blockade. Although STING agonists have failed clinical trials, precision approaches targeting restoration of tumor cell STING expression have yet to be explored. Here, we report that head and neck squamous cell cancer (HNSCC) exhibits a mechanism of STING suppression related to upregulation of protein tyrosine phosphatase non-receptor (PTPN) type 2 (PTPN2) that is also evident in other cancers. PTPN2 inhibition (PTPN2i) increases HNSCC tumor cell STING by restoring IFNγ-STAT1-mediated induction of STING mRNA. This restores sensitivity to STING agonism and natural killer cell activation, suppressing tumor growth in an immune cell-dependent manner in anti-PD-1 refractory syngeneic HNSCC mouse tumor models in female mice. Together, these findings demonstrate that PTPN2i can unleash STING agonist response, providing a rationale for the evaluation of this therapeutic combination in HNSCC and potentially other cancer types.

Abstract RAS and MAPK pathway alterations are frequent in many histologic subtypes of gynecologic cancers. Promising clinical efficacy has been reported with the combination of avutometinib (RAF-MEK clamp) and defactinib (FAK inhibitor) in KRAS-mutant low-grade serous ovarian cancer, and the combination of trametinib (MEK inhibitor) and ABT-263 (BCL-XL inhibitor) in gynecologic cancers (endometrial, ovarian, cervical). However, novel approaches to RAS inhibition have not yet been fully exploited in the treatment of gynecologic malignancies. The purpose of this study was to analyze RAS alterations in an institutional cohort of gynecologic cancers, and to establish patient-derived models of RAS-mutant rare gynecologic cancers in which to evaluate RAS targeted therapies. Targeted next-generation sequencing of an institutional cohort identified RAS gene alterations in all subtypes of gynecologic cancers. RAS mutations (KRAS, NRAS, or HRAS) were identified in approximately 4000 samples, including 11% of ovarian cancers (KRAS G12A, C, D, R, S, V, G13C, D, others; NRAS hotspots), 26% of uterine epithelial cancers (KRAS G12A, C, D, V, G13C, D, other; HRAS and NRAS hotspots), 10% of cervical cancers (KRAS G12C, D, V), 10% of vaginal cancers (KRAS G12A, V; NRAS hotspots), and 6% of vulvar cancers (HRAS). KRAS amplifications were present in about 3.5% of ovarian cancers and 1% of endometrial cancers. Mucinous ovarian cancers demonstrated the highest frequency of KRAS alterations, occurring in at least 67% of cases. We prospectively collected fresh tissue samples from surgical resections of rare gynecologic cancers and attempted patient-derived organoid (PDO) and/or patient-derived xenograft (PDX) generation from selected cases. As proof of concept, we successfully established a PDO and a PDX model from a KRAS G12D mutated mucinous ovarian cancer, which were sustained for multiple passages in vitro or in vivo. The PDO cells resembled the original patient tumor morphologically and harbored the same mutational profile including KRAS G12D, as confirmed by next-generation sequencing. PDO cells were propagated for multiple passages with maintenance of the KRAS G12D mutation as assessed by digital droplet PCR. Response of the KRAS G12D PDO model to a panel of KRAS targeted agents was assessed using a microfluidic device and fluorescent staining for live, dead, and apoptotic cells. Six-day treatment with MRTX1133 (KRAS G12D inhibitor), AMG 410 (pan-KRAS inhibitor), RMC-6236 (daraxonrasib, RAS (ON) inhibitor), or RMC-9805 (zoldonrasib, RAS (ON) G12D selective inhibitor), each at 100nM, resulted in 70-95% growth inhibition. In conclusion, leveraging the substantial frequency of RAS alterations in rare gynecologic cancers, we have established PDO and PDX models of a KRAS G12D mutated ovarian cancer and demonstrated ex vivo sensitivity to a panel of KRAS inhibitors with different mechanisms of action. Targeting RAS may be a promising treatment approach in gynecologic cancers. Citation Format: Elizabeth H. Stover, Magdalena Zielinska, Minh Ha, Satyakam Mishra, David L. Kolin, Cam Anh Tran, Cloud P. Paweletz, Andrew J. Aguirre, Guruprasad Ananda, Elena Ivanova, Ursula A. Matulonis, Joyce F. Liu. Targeting RAS in gynecologic cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr A038.

In estrogen receptor-positive (ER +) breast cancer, CDK4/6 inhibitors (CDK4/6is) combined with endocrine therapy (ET) are standard first-line treatment for metastatic disease. However, most patients eventually develop resistance. Activating ESR1 mutations are a prevalent mechanism of acquired resistance to ET and are enriched after ET plus a CDK4/6 inhibitor (CDK4/6i), but their role in the clonal evolution and adaptive mechanisms of acquired resistance to CDK4/6 inhibition, independent of ET, is unknown. In addition, whether different CDK4/6is impose distinct selective pressures and divergent resistance states remains elusive. To investigate the clonal dynamics, cell states and cellular plasticity during acquired CDK4/6i resistance in mutant versus wild-type (WT) ESR1, we performed high-complexity DNA barcoding (ClonTracer library) with longitudinal sampling and multi-omic profiling in an isogeneic MCF7 model expressing WT ER or Y537S mutant ER. We also evaluated the clonality of the ESR1 mutations in clinical samples with CDK4/6i resistance. We showed that ESR1 mutations are enriched in clinical tumors with acquired resistance to CDK4/6is, and in paired biopsies expanded to near clonality after treatment. We demonstrated progressive clonal selection with both divergent and partially convergent evolutionary trajectories. The ESR1 mutation substantially reshapes clonal and epigenetic evolution during palbociclib resistance but had a weaker impact under abemaciclib selection. Overall, clonal evolution and cell states in palbociclib and abemaciclib resistance were distinct. Single-cell RNA-seq revealed transcriptional heterogeneity highlighting cellular plasticity during passaging of cells and selection. Finally, in vivo barcoding of mammary xenograft, local recurrences, and distant metastases demonstrated site-specific clonal outgrowth in mutant ER metastases, and partial overlap between metastatic and CDK4/6i-resistant subclones, supporting the dual role of specific populations in therapeutic resistance and metastatic colonization. High-resolution lineage tracing and multi-omic studies demonstrate that CDK4/6i resistance is shaped by clonal selection and adaptive remodeling of cell states, with the ESR1 mutation status and the specific inhibitor acting as key determinants of evolutionary trajectories. These findings suggest that both variables should be considered when designing sequential and combination treatment strategies to overcome CDK4/6i resistance.

Abstract Background: High-grade endometrial cancers (EC), including serous, carcinosarcoma, and clear cell subtypes, frequently harbor TP53 mutations, replication stress (RS), and cell-cycle dysregulation, suggesting vulnerability to WEE1 inhibition. We conducted a translational study to define RS-linked pharmacodynamics and ex vivo correlates of response in high-grade EC PDOs. Methods: PDOs from high-grade EC biopsies and surgical samples were treated ex vivo with azenosertib (200 nM, 24 h). DNA-fiber assays measured: (i) fork speed (CldU+IdU as ongoing replication forks), (ii) replication fork stability under RS caused by hydroxyurea (HU), using the IdU:CldU ratio after WEE1i+HU, and (iii) single-stranded (ss)DNA gap formation via the S1 nuclease assay (WEE1i±S1). Results: Four PDOs were profiled: DF4850 and DF4161 (WEE1i-sensitive; IC50s<100nM) and DF042 and DF4968 (insensitive; IC50s>1500nM). Across all four models, fork speed decreased after azenosertib, consistent with on-target effects on RS. However, fork degradation and gap formation aligned with viability. Specifically, under WEE1i+HU, the sensitive models (DF4850, DF4161) showed a significant reduction in the IdU:CldU ratio, indicating impaired fork restart/stability. The insensitive models (DF042, DF4968) were able to protect forks from degradation despite WEE1 inhibition and showed no change. In the S1 gap assay, DF4850/DF4161 exhibited shorter fiber tracts with WEE1i+S1 vs WEE1i alone, consistent with increased ssDNA gaps, whereas DF042/DF4968 again showed no difference, indicating the ability to tolerate WEE1i-induced stress. Thus, while fork slowing is a uniform pharmacodynamic effect of WEE1 inhibition, HU-sensitive fork instability and S1-sensitive gap formation are enriched in sensitive PDO models. Conclusions: In high-grade EC PDOs, azenosertib uniformly slows replication forks, but fork stability under HU and S1-detectable gap formation distinguish WEE1i-sensitive (DF4850, DF4161) from insensitive (DF042, DF4968) models. These mechanism-anchored DNA-fiber readouts merit prospective evaluation as predictive and pharmacodynamic biomarkers for WEE1 inhibitors in high-grade EC. Additional biomarker analyses, including immunohistochemical analyses of RS proteins, are ongoing and will be presented. Citation Format: Elena Ivanova, Ke Cong, Shrabasti Roychoudhury, David Han, Magdalena Zielinska, Minh Ha, Abrielle Jens, Vaishnavi Anand, Bose S. Kochupurakkal, Courtney H. Qi, Arunika Shee, Maureen Mulready, Madison Zizzo, Alexis Rabbitt, Jennifer D. Curtis, Nabihah Tayob, Marisa R. Nucci, Cam A. Tran, Panagiotis A. Konstantinopoulos, Geoffrey I. Shapiro, Dipanjan Chowdhury, Cloud P. Paweletz, Ursula Matulonis, Joyce Liu. DNA fiber-based replication phenotypes distinguish WEE1-inhibitor response in high-grade endometrial cancer patient-derived organoids (PDOs) treated with azenosertib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 243.

Figure S4 shows TREX1 depletion increases sensitivity of resistant SCLC cells to chemotherapy

Small cell lung cancer (SCLC) transformation is an incompletely characterized mechanism of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in EGFR-mutant cancers, limiting development of optimal treatment approaches. Through single-cell RNA sequencing of malignant pleural effusions from patients who underwent SCLC transformation, we identified heterogeneity and diversity, including distinct neuroendocrine (NE) and mesenchymal non-NE cancer cell subsets, which were maintained in patient-derived cell lines. We demonstrate that EZH2 regulates EGFR expression in NE cells where EGFR expression is silenced at baseline. Although neither epigenetic derepression nor exogenous overexpression of mutant EGFR sensitized the cells to EGFR inhibition, non-NE cells exhibited selective sensitivity to MEK inhibitors. Combined MEK inhibitor and chemotherapy effectively inhibited growth of both NE and non-NE cells in vitro and in vivo. Our findings demonstrate that EGFR-mutant SCLC is composed of mixed cell states with distinct therapeutic vulnerabilities and offer a therapeutic strategy to target tumor heterogeneity in highly plastic and treatment-resistant malignancies such as transformed SCLC.

Aging is a major risk factor for breast cancer, yet how it shapes tumor development, molecular phenotype, and immune evasion remains incompletely understood. Deciphering how aging influences cancer evolution is critical for improving risk assessment, prevention, and treatment. Here, using a N-nitroso-N-methylurea (NMU)-induced rat mammary tumor model that recapitulates key features of human breast cancer, we integrated bulk and single-cell transcriptomics, whole-exome sequencing, and histopathological analysis to dissect age-associated differences in mammary tumorigenesis. We found that the age at NMU exposure critically influences tumor incidence, mutational burden, molecular subtype, and the tumor immune microenvironment. Tumors arising in aged rats originated from aging luminal progenitor-like cells, exhibited increased genomic instability, reduced immune cell infiltration, and impaired antigen presentation linked to loss of heterozygosity at chromosome (Chr) 20p. The age-associated epithelial and immune changes we identified were conserved in human breast cancers, where the loss of the homologous Chr 6p region correlated with reduced lymphocyte infiltration and shorter relapse-free survival. These findings reveal that aging profoundly affects tumor-initiating cell populations and promotes immune evasion through chromosomal instability-driven defects in antigen presentation. Our work provides a molecular basis for understanding disease onset and progression that may impact efficacy of immunotherapy in older breast cancer patients.

Abstract Background: Datopotamab deruxtecan (Dato-DXd, DATROWAY) is a TROP2-directed ADC with a highly potent Topo I inhibitor payload, which is approved for HR+ breast cancer and EGFR-mutated NSCLC in the US and is being investigated in several registrational phase 3 trials, including 1L NSCLC, 2L+ TNBC, and urothelial carcinoma. While the cytotoxic payload-induced apoptotic effect of DXd is well investigated, its immunomodulatory effect is less characterized. Here we analyze tumor cell-intrinsic immunological response to Dato-DXd using short-term microfluidic culture of patient-derived organotypic tumor spheroids (pDOTS) in non-small cell lung cancer (NSCLC) samples. Methods: Four surgical NSCLC cases collected from Brigham and Women’s Hospital under an IRB-approved protocol were studied. TROP2 antigen density on EpCAM+ cells was assessed by quantitative flow cytometry and immunofluorescence; baseline immune profile was assessed by flow. Ex vivo response to vehicle, unconjugated Datopotamab, IgG-DXd, and Dato-DXd were assessed by live/dead imaging endpoint analysis. Modulation of tumor cell immunogenicity at 24hr and 72hr was analyzed by 10x single cell RNA sequencing (scRNAseq). Results: Four NSCLC explants with high tumor cell content and high TROP2 expression (3+ by IF) were studied. Response to drug treatment was measured by change in raw live cell area compared to control samples. Unexpectedly, EpCAM+TROP2+ tumor cells from all four samples at both timepoints exhibited negative pathway enrichment scores for Hallmark TNF-α Signaling via NFκB and Inflammatory Response when comparing Dato-DXd to control-treated. Individual gene level analysis revealed significant downregulation of myeloid-associated cytokines (IL-23a, IL-6) and chemokines (CSF1/2, CXCL1/2/3, CCL2, CCL20, IL-8), indicating possible reduced recruitment of myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Investigation of tumor cell immunogenic cell death in response to Dato-DXd treatment revealed inconsistent induction of pro-inflammatory signaling. Conclusions: Our results indicate clear and consistent immunomodulation of NSCLC tumor cells when treated with Dato-DXd, principally downregulation of myeloid-associated inflammation. Statistically significant differential expressions of CSF1/2, CXCL1/2/3, and IL-8 (CXCL8) chemokines reveal a potentially unappreciated mechanism underlying ADC therapeutic efficacy: reduced recruitment of pro-tumoral myeloid cells by tumor cells. Citation Format: Satoru Yasuda, Patrick Hall Lizotte, Elena Ivanova, Zhaorong Li, Yui Tanaka, Daisuke Okajima, Minh Ha, David A. Barbie, Cloud P. Paweletz. Immunomodulatory response in Dato-DXd-treated non-small cell lung cancer patient-derived organotypic tumor spheroids (pDOTS) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2830.

Stimulator of interferon genes (STING) has emerged as a critical cancer immunotherapy target. However, no STING agonist has advanced beyond phase I/II clinical trials, as obstacles center around applying STING agonism to the appropriate clinical context, retaining it in the tumor microenvironment (TME), and limiting its T cell toxicity. Using triple-negative breast cancer (TNBC), we identify defective STING turnover as a cancer state promoting hypersensitivity to STING agonism. We also repurpose a US Food and Drug Administration (FDA)-approved polyethylene glycol (PEG) biopsy marker to deliver STING agonists in a controlled release fashion into the TME. However, STING agonist-induced T cell toxicity limits robust endogenous clonal T cell response, which can be overcome by sequential co-delivery of the STING agonists with CAR T cell therapy using the same PEG marker, eradicating orthotopic TNBC in mouse models while also controlling distant disease. These findings identify a highly translatable platform to combine STING agonists with CAR T cell therapy locally for TNBC and potentially other solid cancers.

PURPOSE:We previously developed quality control materials (QCMs) to aid in the development of circulating tumor DNA (ctDNA) assays. In this study, we further characterize the performance of the QCMs relative to clinical samples. METHODS:QCMs were provided by three manufacturers. To functionally characterize the QCMs, we (1) evaluated EGFR L858R and ex19del (range, 0.5%-5.0% variant allele frequency [VAF]) in QCMs compared with clinical samples by droplet digital polymerase chain reaction (ddPCR), targeted-amplicon sequencing (Tag-seq), and hybrid capture next-generation sequencing (NGS); and (2) evaluated the QCMs and clinical samples near the Tag-seq limit of detection. A clinical pilot was also conducted in 11 clinical laboratories spanning four continents. RESULTS:For functional characterization, part 1, QCM VAFs for hybrid capture were similar to ddPCR for EGFR L858R but lower for ex19del. By contrast, hybrid capture results for EGFR L858R clinical samples showed a positive trend compared with ddPCR. For amplicon NGS, QCMs performed similarly to clinical samples for both variants. For part 2, observed hit rates approximated expected values. In the clinical pilot, median ex19del VAF was higher for Tag-seq than hybrid capture for both 1.0% and 0.5% QCM formulations. Median QCM L858R VAFs were similar for Tag-seq and hybrid capture, with greatest interlaboratory differences observed for Thermo Fisher Scientific QCMs. For non-EGFR variants, we observed assay and QCM-dependent trends, with no particular QCM or assay driving these trends. CONCLUSION:This project revealed unexpected differences in performance of both assays and QCMs. These findings highlight the need for further validation across diverse alteration types and merit consideration by laboratories that rely on QCMs to develop and perform ctDNA assays for diagnostic applications.

PURPOSE:Next-generation sequencing assays for ctDNA analysis are routinely used in the care of patients with advanced non-small cell lung cancer. However, variable assay sensitivities in detection of fusions have been reported. Here, we report on the performance of detecting RET rearrangements in plasma across three commercial next-generation sequencing laboratories. EXPERIMENTAL DESIGN:Banked plasma from the phase 3 LIBRETTO-431 trial was studied. For each patient (n = 60) with a known RET fusion by local tumor tissue genotyping, pretreatment plasma was divided into two 3-mL aliquots and tested on two of three: Guardant Health's Guardant360, Foundation Medicine's FoundationOneLiquid CDx, and Resolution Bioscience's ctDx-First. A round-robin comparison was performed across vendors using three pairwise comparisons of 20 patients each. On an exploratory basis, agreement of fusion breakpoint calling between plasma and tissue and determinants of false negatives in plasma were assessed. RESULTS:Of 40 samples received by each laboratory, 100% (40/40), 92.5% (37/40), and 90% (36/40) were successfully sequenced by Guardant360, FoundationOne Liquid CDx, and ctDx-First, with a RET fusion or rearrangement detected in 60% (24/40), 63.9% (23/36), and 67.6% (25/37) of cases, respectively. Discordant results included rare and common RET translocations but were usually below allelic frequency of 0.5%. Of samples with a RET fusion detected in plasma and a reported fusion partner by tumor assay, the same fusion partner was identified in tissue and liquid 81% to 89% of the time. CONCLUSIONS:Our results support the utility of ctDNA assays concurrently with tissue testing for detection of translocations, with opportunities to further optimize performance. See related commentary by Davies, p. 2264.

Ovarian clear cell carcinoma (OCCC) is a rare subtype of ovarian cancer, often resistant to platinum-based chemotherapy. OCCC is characterized by ARID1A mutations, PI3K and RAS/MAPK pathway alterations, and activation of the HIF signaling pathway. We and others showed that OCCC are sensitive to inhibition of BCL-XL, a pro-survival protein in the intrinsic pathway of apoptosis. Patient-derived models of OCCC could potentially accelerate pre-clinical testing of therapeutics in this difficult-to-treat cancer, but relatively few patient-derived organoid (PDO) or xenograft models of OCCC have been reported. We describe the establishment and propagation of two PDO models of OCCC. Patient 1 had recurrent OCCC with PIK3CA mutation and HER2 amplification. Patient 2 had recurrent OCCC with CDKN2A/B deletion. Both patients underwent secondary cytoreductive surgery for recurrent disease. Fresh tissue was collected under IRB-approved protocols and tumor cells were plated in Matrigel and formed PDOs. Patient 1 OCCC cells have been propagated for over 35 passages, and patient 2 cells for 9 passages to date. Immunohistochemistry to confirm fidelity to the original tumor is in progress. As a proof-of-concept for evaluating therapeutics in OCCC PDOs, we used a 3D microfluidic device (AIM Biotech) to evaluate drug responses in patient 1 PDO. PDO cells were seeded in the microfluidic device and expanded for one week, treated with drugs within the device, then stained at day 6 with fluorescence markers for live (TMRM, Thermo Fisher), apoptotic (CellEvent Caspase-3/7, Thermo Fisher), and dead (DRAQ7, CST) cells. Quantitative imaging was performed to determine the cell area positive for each marker. The OCCC PDO was treated with standard-of-care chemotherapies carboplatin and paclitaxel; BCL-XL inhibitor A1331852 (Selleckchem); a novel BCL-XL PROTAC degrader DT2216 (Dialectic Therapeutics); or combinations of the BCL-XL inhibitor/degrader with paclitaxel. Treatment with A1331852 or DT2216 induced apoptosis and death in the OCCC PDO cells compared to control. While paclitaxel alone also induced some cell death, the combination of DT2216 (1µM) and paclitaxel (500nM) was highly potent, resulting in elimination of almost all the OCCC cells (Bliss synergy score 0.28±0.08, p=0.013). These data suggest that BCL-XL inhibition/degradation combined with paclitaxel may be a promising treatment strategy for OCCC. Our study also demonstrates the successful establishment of OCCC PDOs and application of a microfluidic device for evaluating novel therapeutic strategies in ovarian cancer. Clare E. Padrick, Magdalena Zielinska, Minh Ha, Aisha L. Saldanha, Cam Anh Tran, Brendan Shay, Daohong Zhou, Kristopher A. Sarosiek, Cloud P. Paweletz, Ursula A. Matulonis, Joyce F. Liu, Elena V. Ivanova, Elizabeth H. Stover. Organoid models of ovarian clear cell carcinoma for evaluating therapeutic sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 40.

Figure S12. In vivo kinetics of CAR infiltration into PC9 subcutaneous tumor. PC9 tumors once established at ∼100 mm3 were injected with either 2.5E6 irrelevantly targeted control or TROP2 CAR+ cells expressing NanoLuciferase, which allows for in vivo tracking of CAR. Mice were imaged after injecting with FFz substrate at indicated time points post CAR engraftment, with right flank representing PC9 mCRD and left flank PC9 mCPD tumor inoculation site.

BCMA and TROP2 CAR-T cell therapies efficacy versus sacituzumab govitecan in the DFCI-243 PDX model.

Figure S9. Phenotyping of CAR T cells. A. CAR T were generated and on day 5 post transduction, cells were washed with FACS buffer, and stained with antibodies as indicated. A. CD4+ and CD8+ populations in different CAR T cell groups B. Assessment of T-cell memory of CAR T products via staining of CCR7 and CD45RA across the indicated conditions. Data are representative of independent experiments with 2 different donors.

Figure S1. TROP2 CAR T construct design and specificity. A. Second-generation TROP2 CAR construct design. scFv was derived from Sacituzumab. Expressed in lentiviral vector with CMV promoter, IgG Kappa signal peptide, CD28 transmembrane domain (TM), and 4-1BB and CD3z costimulatory domain followed by P2A and VexGFP. B. Cytotoxicity assay of TROP2 CARs against PC9 WT (EGFR exon 19 del) and PC9 clone with knockout of TROP2. Cells were incubated with E:T of 1:1 and incubated for 24 h before cell viability of PC9 was read. Viability reported normalized to irrelevant control CAR. C. In vitro cytotoxicity of HCC827GR6 and (NSCLC EGFR exon 19 del/MET amp) and MDA-MB-231 (TNBC). P values reported as follows compared to control: ns, P > 0.05; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001