Immunotherapy Resistance Research Articles

Abstract P3-06-27: Immunotherapy and PI3K/mTOR inhibition combination to mediate metastasis and immunotherapy resistance in triple-negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) tends to be highly aggressive, and almost 50% of patients develop distant metastasis to the brain, liver, and/or lung, at which point the median survival time is only 8-13 months. The average time to relapse in TNBC patients is 19-40 months and the mortality rate within 3 months after recurrence is 75%. Although the addition of immunotherapy to neoadjuvant chemotherapy has improved the survival of patients with stage II–III TNBC, drug resistance remains a problem. Hence there is an urgent need to develop new therapy regimens and targets. Methods: Extensive preclinical program initially starting with in vitro breast cancer cell lines (MDA-MB-231 and MCF-7) to evaluate various PI3K and PI3K-mTOR inhibitors using WST-1 cell proliferation assays, cell migration assays, qRT-PCR and immunofluorescence analysis. In vivo studies utilised the highly aggressive, immunotherapy resistant, TNBC mouse model, 4T1. Briefly, six-to-eight-week-old female BALB/c mice were inoculated with 1 x 105 4T1 cells administered into the mammary fat pad. Once tumours reached ∼50-100 mm3, the PI3K-mTOR inhibitor, Paxalisib, was administered daily by oral gavage in combination with either anti-PD-1 (pembrolizumab) treatment or PARP inhibitor (olaparib). Extensive pathology, nanostring and CODEX spatial analysis was used to evaluate primary tumour burden, metastases and inflammation. Results: Dual targeting of the PI3K-mTOR pathway but not PI3K alone inhibited cancer cell proliferation and migration and promoted a favorable mesenchymal-to-epithelial phenotype. In vitro, the PI3K-mTOR inhibitor, Paxalisib, reduced metastasis-initiating cell signatures including the highly aggressive CD44high/CD24low CSC phenotype, persister cancer cell phenotype (p65, FOXQ1, NRF2, NNMT), and drug resistance markers (ABCB5, SNAIL, ALDH1). PI3K-mTOR blockade also reduced NFκB p50 and the pro-inflammatory downstream target IL-6 whilst inducing viral mimicry genes, making cancer cells more immune visible. Dose de-escalation of paxalisib in the highly aggressive 4T1 TNBC mouse model substantially reduced primary tumor burden, lung metastases, and liver inflammation in combination with immunotherapy +/- chemotherapy whilst overcoming toxicity complications and resistance associated with standard-of-care treatment. Paxalisib in combination with the PARP inhibitor olaparib also reduced primary tumor burden and metastases compared with olaparib monotherapy. NanoString analysis of paxalisib +/- immunotherapy-treated tumors showed a marked reduction in “pro-tumorigenic” immune populations whilst CODEX spatial analysis revealed an increase in the infiltrating adaptive immune arm following paxalisib + anti-PD-1 treatment. Conclusions: The addition of paxalisib to immunotherapy in the 4T1 TNBC mouse model reduced primary tumor burden, lung metastases, and liver inflammation whilst overcoming toxicity complications and resistance associated with standard-of-care immunochemotherapy. Paxalisib in combination with the PARP inhibitor olaparib, but not monotherapy alone, also reduced primary tumor burden and metastases. Moving forward, work is ongoing to elucidate the PI3K-mTOR mechanism of overcoming metastasis and drug resistance as well as the translation of the data into a clinical development program for patients with TNBC and advanced breast cancer. Citation Format: John Friend, Michelle Melino, Wen Juan Tu, Taniya Ahuja, John Vandermeide, Martina Proctor, Amanda Bain, Gahyathiri Nallan, Helle Bielefeldt-Ohmann, Sudha Rao. Immunotherapy and PI3K/mTOR inhibition combination to mediate metastasis and immunotherapy resistance in triple-negative breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-06-27.

Overcoming acquired immunotherapy resistance in non-small cell lung cancer using ginsenoside Rb1-loaded, peptide-enhanced exosome delivery systems

Non-small cell lung cancer (NSCLC) remains a major global health challenge, with limited therapeutic success due to drug resistance and the immunosuppressive tumor microenvironment (TME). This study explores a novel strategy to overcome acquired resistance to immunotherapy in phosphoinositide 3-kinase (PI3K)-mutated NSCLC. Tumor-derived exosomes (T-exo) were modified with the tumor-targeting peptide TMTP1 and loaded with Ginsenoside Rb1 (Rb1) via electroporation to develop peptide-modified Rb1@T-exo. This innovative delivery system demonstrated enhanced tumor-targeting ability and improved stability and bioavailability of Rb1. Both in vitro and in vivo experiments revealed that Rb1@T-exo effectively suppressed tumor growth and metastasis, significantly inhibited the PI3K/AKT/mTOR signaling pathway, and remodeled the immune microenvironment by promoting M1 macrophage polarization and enhancing CD8+ T cell proliferation and cytotoxicity. Transcriptomic and bioinformatic analyses identified key differentially expressed genes (DEGs) and pathways associated with resistance reversal, including the PI3K/AKT/mTOR and PD-1/PD-L1 pathways. Moreover, Rb1@T-exo synergized with immune checkpoint blockade therapy, demonstrating potential as a dual therapeutic approach. This study highlights the potential of peptide-modified Rb1@T-exo as a targeted therapeutic platform for overcoming immunotherapy resistance in PI3K-mutated NSCLC and provides a promising direction for future anti-tumor therapies.Graphical abstract

Oxidized Low-Density Lipoprotein as a Potential Target for Enhancing Immune Checkpoint Inhibitor Therapy in Microsatellite-Stable Colorectal Cancer.

Oxidized low-density lipoprotein (oxLDL) exhibits differential expression in microsatellite-stable (MSS) and microsatellite instability-high (MSI) colorectal cancer (CRC), highlighting its potential therapeutic role in immune checkpoint inhibitor (ICI) resistance in MSS CRC. Elevated oxLDL levels in MSS CRC contribute to tumor progression and diminish ICI efficacy by modulating metabolic reprogramming and immunosuppressive mechanisms within the tumor microenvironment (TME) by activating receptors such as LOX-1 and CD36. oxLDL triggers signaling pathways, including NF-κB, PI3K/Akt, and MAPK, leading to the expansion of immunosuppressive cells like regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and M2 macrophages, while concurrently suppressing effector T cell functions. Additionally, oxLDL enhances oxidative stress and promotes fatty acid oxidation (FAO) and glycolytic metabolism, resulting in nutrient competition within the TME and establishing an immunosuppressive milieu, ultimately culminating in ICI resistance. This review systematically examines the disparities in oxLDL expression between MSS and MSI CRC and elucidates the molecular mechanisms through which oxLDL mediates ICI resistance. Furthermore, it explores potential therapeutic strategies targeting oxLDL, offering novel avenues to overcome immunotherapy resistance in MSS CRC.

Tumor cell-intrinsic Decr2 regulates ferroptosis and immunotherapy efficacy.

Immune checkpoint blockade therapies have transformed the landscape of cancer care, but durable clinical responses are achieved in only a subset of patients. To identify genes that can contribute to immunotherapy resistance, a genome-wide CRISPR screen was performed. Selection for mutants resistant to T cell-mediated killing identified the gene encoding Decr2, a peroxisomal 2,4-dienoyl-CoA reductase. We show that Decr2 in tumor cells participates in CD8+ T cell-mediated tumor cell killing and that Decr2 knockdown reduces the efficacy of anti-PD-L1 therapy in vivo. Knocking down Decr2 expression resulted in diminished ferroptosis which was associated with reduced induction of polyunsaturated ether phospholipids (PUFA-ePLs). Analysis of tumor RNAseq data from human melanoma patients revealed that up-regulation of Decr2 was associated with anti-PD-1 efficacy, and patients with Decr2 gene deletions showed worse clinical outcomes. Our results identify Decr2 as a regulator of immune-mediated tumor cell killing, with implications for improving immunotherapy efficacy.

Acquired resistance to immunotherapy by physical barriers with cancer cell–expressing collagens in non–small cell lung cancer

Immunotherapy has become the standard treatment for many types of cancers, but an increasing number of patients who initially respond to these treatments develop acquired immunotherapy resistance (AIR). Here, we recapitulated the entire process of immunotherapy from response to AIR in mice with non-small cell lung cancer (NSCLC). With implanted tumor organoids derived from these models and serial transplants, we demonstrated that tumor cell-intrinsic mechanisms contributed significantly to AIR. Single-cell RNA sequencing and electron microscope assays revealed that resistant tumor cell-expressing collagens, including Col3a1 and Col6a1, formed multiple physical barriers surrounding tumor cells. Disruption of these barriers by collagenase or knockout of both Col3a1 and Col6a1 in tumor cells could sensitize the tumors of AIR. Mechanistically, the TGFβ pathway was upregulated upon immunotherapy, and treatment with TGFβ significantly increased the expression levels of both Col3a1 and Col6a1 in tumor cells. COL3A1 formed a castle-like barrier for a cluster of tumor cells and prevented T cell infiltration, while COL6A1 formed an armor-like barrier surrounding individual tumor cells to protect them against direct T cell attack. Our data reveal a tumor cell-intrinsic mechanism of AIR, mediated by collagen-containing physical barriers, which immediately suggests a clinical treatment option.

NGR-modified nanovesicles target ALKBH5 to inhibit ovarian cancer growth and metastasis

Background: Immunotherapy resistance in ovarian cancer (OC) poses a significant clinical hurdle. This study aims to investigate the potential of NGR-modified biomimetic nanovesicles (NGR-BNVs) for delivering ALKBH5 siRNA to reverse this resistance.Methods:In vitro and in vivo experiments were conducted to assess the efficiency of NGR-modified nanovesicles in delivering ALKBH5 siRNA. OC cell proliferation was evaluated, and apoptosis induction was measured. A mouse xenograft model was utilized to examine the effects on tumor volume and metastasis. Tumor immune microenvironment (TIME) analysis was performed to determine changes in immune cell proportions and immunomodulatory factors.Results: NGR-modified nanovesicles effectively delivered ALKBH5 siRNA, leading to a significant inhibition of OC cell proliferation and apoptosis induction. Treated groups in the mouse xenograft model exhibited reduced tumor volume and decreased metastatic signals. Analysis of the immune microenvironment revealed an increased proportion of CD8+ T cells, reduced Tregs and MDSCs, and notable changes in key immunomodulatory factors.Conclusion: This study highlights the potential of NGR-modified BNVs for overcoming immunotherapy resistance in OC by delivering ALKBH5 siRNA, resulting in modulation of the immune microenvironment and promising therapeutic outcomes.

Glutamate transporter SLC1A6 promotes resistance to immunotherapy in cancer

BackgroundResistance to immune checkpoint inhibitors remains a significant challenge in the treatment of cancer. Emerging evidence suggests that metabolic reprogramming plays a crucial role in tumor metabolism and progression. Our study strived to investigate the role and underlying mechanisms of the glutamate transporter SLC1A6 in resistance to immunotherapy of cancer.MethodsSingle-cell RNA sequencing was performed on bladder cancer patients receiving neoadjuvant immunotherapy to identify the expression of SLC1A6 in treatment-resistant cases. The clinical prognostic value of SLC1A6 in cancer was validated using publicly available lung cancer single-cell datasets, as well as transcriptomic data from both bladder and lung cancer cohorts. Flow cytometry was employed to assess the impact of SLC1A6 knockdown on the effector function of CD8⁺ T cell. In vivo tumor models were used to evaluate the role of SLC1A6 in immunotherapy resistance, with immunofluorescence staining performed to examine GZMB⁺ CD8⁺ T cell infiltration.ResultsSLC1A6 was highly expressed in bladder cancer patients resistant to neoadjuvant immunotherapy, and its expression was associated with disease progression, poor prognosis, and low immune infiltration. Knockdown of SLC1A6 in tumor cells enhanced CD8⁺ T cell effector function. SLC1A6 knockdown also improved the efficacy of immunotherapy and increased the infiltration of GZMB⁺ CD8⁺ T cells within the tumor microenvironment.ConclusionsSLC1A6 plays a critical role in resistance to immunotherapy in cancer. Targeting SLC1A6 may provide a promising therapeutic strategy for improving responses to neoadjuvant immunotherapy and advancing combination treatment approaches.

Starting the Engine and Releasing the Brakes of T-Cell Responses: A Biomimetic Dendritic Cell Nanoplatform for Improved Glioblastoma Immunotherapy.

Glioblastoma (GBM), the most aggressive primary brain tumor, remains a challenge for immunotherapies, like immune checkpoint blockade (ICB), due to the blood-brain barrier (BBB) and immunosuppressive tumor microenvironment (TME) deficient in cytotoxic T-cells and effective T-cell-dendritic cell (DC) interactions. Herein, we engineer a biomimetic nanoplatform comprising paclitaxel (PTX) nanoparticles (NPs) encapsulated in a tumor-associated antigen-loaded DC membrane modified with ICB antibodies. The DC membrane not only facilitates BBB penetration and GBM targeting but also directly engages with T-cells reminiscent of T-cell-antigen-presenting cell (APC) clusters. Simultaneously, PTX NPs induce immunogenic cell death, eliciting persistent stimulatory signals for DC maturation and subsequent T-cell priming, thus synergistically "starting the engine" of T-cell immune responses. Meanwhile, ICB antibodies further "release the brakes" by mitigating T-cell exhaustion and dysfunction. In GBM-bearing mice, this nanoplatform outperformed ICB monotherapy, significantly inhibiting tumor growth and prolonging survival by reshaping the TME. We observed increased number of cytotoxic T-cells proximal to DCs that form T-cell-APC clusters, accompanied by enhanced T-cell proliferation and effector function. This study provides a promising paradigm for overcoming immunotherapy resistance in GBM.

LMNB2-mediated high PD-L1 transcription triggers the immune escape of hepatocellular carcinoma

While immune checkpoint inhibitors targeting programmed cell death-ligand 1 (PD-L1) demonstrate clinical efficacy in hepatocellular carcinoma (HCC), tumor cells frequently evade immune surveillance through PD-L1 overexpression, a phenomenon whose regulatory mechanisms remain poorly understood. Through integrated analysis of single-cell transcription sequence data, we identified aberrant upregulation of Lamin B2 (LMNB2) specifically in immunotherapy-sensitive HCC patients. Functional characterization revealed that LMNB2 acts as a transcriptional regulator of PD-L1, potentiating immune escape mechanisms in HCC cells during co-culture with Jurkat cells. Notably, we discovered that speckle-type POZ protein (SPOP) directly interacts with LMNB2 to mediate its ubiquitination and proteasomal degradation, thereby maintaining physiological PD-L1 expression levels. Clinically relevant SPOP mutations or reduced SPOP expression impaired this regulatory mechanism, leading to LMNB2 accumulation and subsequent PD-L1 hyperactivation. Importantly, combinatorial targeting of LMNB2 with Atezolizumab (PD-L1 inhibitor) displayed a synergistic effect on suppressing tumor progression both in vitro and in vivo, particularly in HCC models with SPOP mutations or LMNB2 overexpression. These findings unveil a novel ubiquitination-dependent regulatory axis in HCC immune evasion and propose targeted co-inhibition strategies to overcome HCC immunotherapy resistance.

Tumor suppressor SLC9A2 inhibits colorectal cancer metastasis and reverses immunotherapy resistance by suppressing angiogenesis

BackgroundColorectal cancer (CRC) is a common and aggressive malignancy that frequently metastasizes to the liver, presenting significant therapeutic challenges. Despite its clinical importance, the mechanisms underlying CRC liver metastasis and resistance to immune therapy remain poorly understood. In this study, we aimed to investigate the molecular mechanisms driving CRC metastasis using a novel approach, which includes the establishment of highly metastatic CRC cell lines.MethodsTo explore the role of specific genes in CRC liver metastasis, we developed two highly metastatic CRC cell lines (LoVo-Hm and HCT116-Hm) by applying sustained selective pressure to primary CRC cells. RNA sequencing was performed to identify differentially expressed genes in these metastatic cells. Additionally, we conducted assays for cell migration, invasion, angiogenesis, and ELISA to evaluate VEGFA production, all to confirm the functional role of SLC9A2. Our findings were further validated in human CRC tissue samples and publicly available datasets to assess the clinical relevance of the identified targets.ResultsOur analysis revealed a significant downregulation of SLC9A2 in the highly metastatic CRC cell lines. Mechanistically, we found that SLC9A2 inhibits epithelial-mesenchymal transition (EMT) and metastasis by suppressing the STAT3 signaling pathway. Moreover, SLC9A2 reduces VEGFA secretion, normalizing tumor vasculature and reshaping the tumor microenvironment (TME), which ultimately enhances anti-tumor immunity. Comparative analysis of CRC tissue samples showed reduced SLC9A2 expression in tumor tissues compared to adjacent normal tissues, with a negative correlation to TNM staging. Importantly, higher SLC9A2 expression was associated with better treatment responses in immunotherapy cohorts.ConclusionThese findings highlight the critical role of SLC9A2 in regulating metastasis, angiogenesis, and TME remodeling in CRC. By modulating the STAT3 pathway and tumor vasculature, SLC9A2 emerges as a potential prognostic biomarker and therapeutic target. Targeting SLC9A2 may enhance immune responses and improve treatment outcomes in CRC, offering a promising avenue for future therapeutic strategies.

Targeting metabolic reprogramming to overcome immune tolerance in melanoma immunotherapy.

Significant advances in the treatment of melanoma, the most aggressive form of skin cancer, have been achieved via immunotherapy. Despite these improvements, therapeutic resistance remains a formidable challenge, compromising the treatment efficacy and patient outcomes. This review delves into the intricate mechanisms driving immunotherapy resistance in melanoma, emphasizing alterations in key metabolic pathways, changes within the tumor microenvironment, and the critical role of the gut microbiota. This review also examines how metabolic reprogramming supports tumor proliferation and immune evasion, it highlights the impact of extracellular acidification and angiogenic processes on resistance development. By synthesizing current insights, this review emphasizes the importance of targeting these multifaceted interactions to overcome resistance, thereby paving the way for more effective and durable therapeutic strategies in melanoma treatment.

Beyond the tumor: the gut microbiome as a key player in immunotherapy efficacy and resistance.

Cancer immunotherapy aims to use the immune system of the body for improved therapeutic effects on tumors. Currently, one of the more encouraging interventions under evaluation involves the use of immune checkpoint blockade, which offers longer benefit periods and greater patient tolerance than previous interventions for solid malignancies. Nevertheless, a majority of patients never respond or gradually acquire resistance; hence, a suboptimal effect of the therapy ensues. Resistance to such treatments may arise from tumor-specific factors, host factors, and environmental influences. There is growing evidence that the gut microbiome is an important modulator not only of the efficacy of these treatments but also of toxicities. Current studies are focused on the identification of key microbial profiles from both preclinical and clinical samples associated with immunotherapeutic response and antitumor activities. Elucidation of this complex interaction may provide ways to modulate gut microbial communities to improve patient outcomes. The current review addresses the components responsible for resistance against immune checkpoint inhibitors and highlights the crucial linkage between gut microbiome-immune interactions. We further summarize some recent clinical findings and explore prospective avenues for research in this evolving area of cancer treatment.

A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

A microfluidic tumor-on-chip platform deciphers hypoxia-driven FOXO3a/PD-L1 signaling in gastric cancer immunotherapy resistance

Regulation of tumor-associated fibroblasts by TACSTD2 in esophageal squamous cell carcinoma and its potential role in immunotherapy resistance.

e16045 Background: Esophageal squamous cell carcinoma (ESCC) has high global incidence and mortality, with poor prognosis due to late diagnosis and limited chemotherapy efficacy. Immune checkpoint inhibitors targeting PD-1/PD-L1 improve survival in some ESCC patients, but benefits are limited to a subset, lacking biomarkers to identify responsive populations. Identifying biomarkers to predict immunotherapy efficacy is crucial for advancing treatment. Methods: Biopsy tissue samples were collected from four ESCC patients before treatment. All patients received three cycles of neoadjuvant therapy with TC regimen plus sintilimab. Post-treatment pathological responses were classified as pCR (pathological complete response), or tumor regression of grades 1 to 3 (P0, P1, P2, and P3). Surgical resection tissue samples were also collected from patients with pCR (P0-after) and tumor regression grade 3 (P3-after). Spatial transcriptomics was performed on these six tissue samples. Additionally, single-cell RNA sequencing data from two untreated patients and one immune therapy-treated ESCC patient from the GSE221561 dataset were included, and cell types were annotated. Results from spatial transcriptomics and single-cell sequencing were analyzed to identify key genes and pathways influencing immunotherapy efficacy, as well as examine cellular distribution, communication, and type-specific effects. The distribution and characteristics of immune cells before and after immunotherapy were explored. Results: (1) Differential genes between non-pCR (P1-P3) and pCR (P0) groups, as well as between Response-good (P0-P1) and Response-poor (P2-P3) groups, included TACSTD2, KRT family, S100A family, membrane proteins, and DSCs. TACSTD2 expression increased across 4 groups, correlating with immunotherapy resistance. Pathway analysis highlighted extracellular matrix organization. (2) Fibroblasts were the second most abundant cell type, with significant differences across 4 groups. These differences matched extracellular matrix-related gene expression, suggesting that tumor-associated fibroblasts (CAFs) influence immunotherapy efficacy by secreting extracellular matrix. (3) TACSTD2 spatial expression significantly overlapped with α-SMA, a CAF marker, suggesting TACSTD2 may induce CAF differentiation. Cell communication analysis showed interactions between ESCC cells and CAFs via TGF-β receptors and ligands. (4) Comparison of non-responding (P3-after) and complete response (P0-after) patients showed differential gene enrichment in extracellular matrix composition and T-cell infiltration, induced by different CAF types. Conclusions: TACSTD2 may regulate CAF differentiation via the TGF-β pathway, promoting immunotherapy resistance through specific extracellular matrix production and T-cell imbalance.

CtDNA-based clinicogenomic analysis of advanced head and neck cancer patients treated with immune checkpoint inhibitors.

6036 Background: Head and Neck cancer (HNC) is among the diverse group of malignancies affecting the head and neck region including the oral cavity. Being the most prevalent malignancy in Southeast Asia, it has a high mortality rate. Despite the advancement in treatment, 5 years survival rate for HNC remains below 50%, and the majority of Pts receiving frontline therapy experience locoregional or incurable metastatic relapse. Immune checkpoint inhibitors (ICI) are recommended for relapsed patients, but only 20% of patients show measurable response. Currently, no predictive biomarkers are available to predict ICI response and there is an urgent need for genomic markers to predict ICI outcomes. Here we report comprehensive genomic profiling (CGP) of advanced HNC patients receiving ICI. Methods: ctDNA from 69 advanced HNC patients receiving combinational immune-chemotherapy were serially profiled at the baseline (BL) and post-treatment (Tx) by targeted, hybridization-based CGP using OncoIndx comprehensive gene panel (CGP) comprising 1080 genes. The ctDNA differential features at BL and post-Tx as well as among responders (R) and non-responders (NR) were correlated with Progression-free survival (PFS) and Overall survival (OS) using Kaplan-Meier statistics and multivariate analysis. Results: Among total patients, 58% (40/69) were responders (R) while the remaining were non-responders (NR). At the population level, HRR pathway tumor suppressors and epigenetic modifiers were the most frequent pathogenic variants. At BL, the NR population was enriched with oncogenic gene mutations compared to the R population. TP53 and BRCA pathway mutations (mTP53 + BRCA) showed a strong association with progression-free survival (PFS) and overall survival (OS). Pts with cooccurring mTP53 + BRCA had significantly lower PFS (median PFS: 2.77 months for mTP53 + BRCA pathway vs 9.1 months for wt TP53 + BRCA pathway. P=<0.0001, HR=3.2-11.6) and OS (median OS: 4.67 months for mTP53 + BRCA pathway vs 12.63 months for wtTP53 + BRCA pathway. P=<0.0001, HR = 11.18-55.27). NOTCH 1 or 2 variants were enriched in R population, with a beneficial effect on survival outcomes. Elevated ctDNA alterations and Tumor fraction (TF) concentrated in the NR population disproportionately contained subclonal potential drivers of immunotherapy resistance including NF1, STAT5 B, and STK11 mutations, and were associated with short survival. Univariate and multivariate analysis suggested that ctDNA mutations, TF, and high mutational heterogeneity emerged as risk factors for shorter PFS and OS. In contrast, total Indel burden and NOTCH mutations had beneficial effects on PFS and OS. Conclusions: Minimally invasive plasma ctDNA CGP showed heterogenous actionable mutations at BL and post Tx and identified immunotherapy resistance conferring genomic markers for stratifying potential responders for immunotherapy guidance.

Application of a novel multiplex imaging-based immunotherapy panel and AI-powered analysis solution for predictive spatial biomarker identification on immunotherapy-treated melanoma patients.

9524 Background: There is an urgent need for more robust methods to differentiate immunotherapy responders from non-responders. In this study, we present a novel multiplex imaging (MI)-based immunotherapy panel and a comprehensive analysis pipeline to characterize the spatial distribution and function of immune cells and its application for spatial biomarker detection in a cohort of immunotherapy-treated melanoma patients. Methods: We designed a 28-plex panel to perform sequential immunofluorescence (seqIF) on the COMET platform to target key biomarkers associated with tumor microenvironment (TME), immune cell infiltration, and immune checkpoint pathways. Pre-treatment biopsies were obtained from 12 patients with known long-term response or rapid progression to immunotherapy combination treatment from the SECOMBIT Trial (NCT02631447) and profiled utilizing Nucleai’s deep-learning-based MI analysis pipeline, aiming to identify spatial biomarkers that can differentiate between long-term responders and non-responders. We identified 15 cell types, including 10 immune cell populations, in addition to 10 cell state markers. Cells were assigned to the tumor area or TME, and spatial features were calculated based on cell type, marker positivity, and gross area assignment. Results: Our novel MI panel and analysis pipeline demonstrated highly balanced accuracy (> 0.8) and F1 scores (> 0.8) in cell typing and protein quantification for most cell types and markers. This analysis pipeline enabled the quantification of known biomarkers such as T cell activation states, T cell infiltration patterns, and tertiary-lymphoid structure maturation. A comparison of calculated spatial features between long-term responders and rapid progressors revealed distinct immune cell interactions and differences in activation status across the tumor areas associated with response. Within the tumor area, the reciprocal interactions of tumor cells, cytotoxic CD8 T-cells and antigen-presenting cells (APC) were associated with a better outcome. In contrast, a high percentage of proliferating regulatory T cells within the tumor invasive margin was associated with a worse outcome. In the adjacent TME, endothelial cell interactions with T-cells and macrophage proliferation were associated with immunotherapy resistance. In contrast, the interaction between HLA-DR-expressing macrophages and APC cells was associated with an improved clinical outcome. Conclusions: Integrating MI with AI analysis has the potential to enhance our understanding of treatment efficacy and resistance mechanisms. Our preliminary data demonstrate that area-specific immune niches contribute to the success or failure of immunotherapy response and highlight the importance of spatial biology in predicting immunotherapy outcomes. Clinical trial information: NCT02631447 .

CCR8 positive Tregs and their correlation with immunotherapy response in advanced non-small cell lung cancer (NSCLC).

2635 Background: Regulatory T cells (Tregs) expressing the chemokine receptor CCR8 are pivotal modulators of the tumor immune microenvironment. CCR8 has recently emerged as a promising therapeutic target due to its selective expression on activated Tregs in the tumor microenvironment and its role in promoting immunosuppression. This study investigates the prognostic and therapeutic implications of CCR8-positive Tregs in non-small cell lung cancer (NSCLC), focusing on their impact in relation to tertiary lymphoid structure (TLS) status. Methods: A validated 6-plex multiplex immunofluorescence (mIF) panel was used to analyze tumor samples from NSCLC patients treated with immune checkpoint blockers (ICB) in the BIP precision medicine study (ClinicalTrials.gov: NCT04389143). Markers included CD4, CD8, CD20, FoxP3, PanCK, and CCR8, alongside DAPI staining to assess immune contexture (infiltrated, excluded, desert), TLS status, and CCR8/FOXP3 double-positive Tregs. Clinical outcomes, including progression-free survival (PFS) and objective response rate (ORR), were analyzed in 50 responders and 50 non-responders. Findings were validated using transcriptomic data from the POPLAR (NCT01903993) and OAK (NCT02008227) studies, which evaluated atezolizumab versus docetaxel in advanced NSCLC. Kaplan-Meier curves, hazard ratios, and Cox regression models were used for survival analyses. Results: CCR8-expressing Tregs were significantly enriched in infiltrated tumors, showing a 1.5-fold increase compared to excluded tumors (p = 0.057), a 3.3-fold increase compared to desert tumors (p = 0.001), and a 1.8-fold increase in TLS-positive tumors compared to TLS-negative tumors (p = 0.003). These findings highlight that activated Tregs co-infiltrate with CD8 T cells and other immune cell types. This enrichment was confirmed in samples from the POPLAR and OAK studies using transcriptomic analyses (3-fold increase, p = 2e-16). Due to their correlation with overall immune cell infiltration, the presence of CCR8-positive Tregs was significantly associated with better survival (HR 0.45, p < 0.001) across the entire patient cohort. However, when stratified for TLS-positive tumors, the presence of activated Tregs was associated with diminished objective response rate and progression-free survival suggesting a negative impact of these immunosuppressive cells on response to ICB. Conclusions: This study provides the first evidence linking CCR8-positive Tregs with immunotherapy resistance in NSCLC, particularly in TLS-positive tumors. These findings parallel observations in TLS-positive sarcomas, where Treg abundance predicted poor outcomes (Italiano et al., Nature Medicine , 2022). This study supports the exploration of CCR8-targeted therapies to deplete immunosuppressive Tregs and enhance the efficacy of immunotherapy in TLS-positive NSCLC.

Delineation of immunotherapeutic predictive versus prognostic transcriptional programs to identify SLC22A5-centric carnitine metabolism-driven resistance to anti-PD-L1 treatment in advanced non–small-cell lung cancer.

2623 Background: Prognostic factors indicate the natural course of a disease regardless of treatment, whereas predictive factors determine the likelihood of response to specific therapies. Distinguishing between predictive and prognostic factors is essential for separating treatment-specific outcomes from the inherent progression of cancer, thereby guiding clinical decision-making. We aim to dissect the predictive and prognostic transcriptional programs underlying the efficacy of anti-PD-L1 versus chemotherapy in advanced non-small cell lung cancer (NSCLC) to uncover mechanisms specific to immunotherapy resistance. Methods: Clinical and baseline tumor transcriptomic data were collected from two randomized controlled trials comparing atezolizumab with docetaxel: OAK (n=697, discovery cohort) and POPLAR (n=192, validation cohort). Transcriptional program scores for each biological process and metabolic pathway from the Reactome database were calculated using gene set variation analysis for each patient. Cox regression and P-value for interaction tests were conducted to differentiate predictive versus prognostic effects of transcriptional programs. Tumor microenvironment and cell-cell communication underlying immunotherapy resistance were explored using bulk and single-cell transcriptomic data. Results: Transcriptional programs in the OAK discovery cohort were divided into four categories associated with different predictive effects specific to atezolizumab or docetaxel. Carnitine metabolism was the most prominent process contributing to atezolizumab-specific resistance, while porphyrin metabolism drove docetaxel-specific resistance. SLC22A5, the only high-affinity carnitine transporter, was upregulated in atezolizumab-resistant patients. The predictive effect of SLC22A5-centric carnitine metabolism for resistance to atezolizumab rather than docetaxel was confirmed in the POPLAR validation cohort. Integrative analyses of bulk and single-cell transcriptomes revealed that cancer cell-specific SLC22A5 expression induced M2 macrophage polarization and decreased CD8+ T cell infiltration via carnitine uptake, thus forming an immunosuppressive microenvironment. Conclusions: Our study elucidates the distinction between predictive and prognostic factors in advanced NSCLC from a metabolic perspective. Cancer cells uptake of carnitine via SLC22A5 mediates resistance to anti-PD-L1 treatment. Combining inhibition of SLC22A5-centric carnitine metabolism with anti-PD-L1 agents might be a promising strategy to reverse immune escape in advanced NSCLC. Keywords: Predictive, Prognostic, Non-small cell lung cancer, Carnitine metabolism, Resistance.

Mechanism study of reversing tumor immune desertification and immunotherapy resistance by low dose radiotherapy.

e15181 Background: The immune classification of tumor can be divided into immune infiltrating type, immune rejection type and immune desert type. There is little infiltration of CD8 + T cells in immune desert tumors, which are less responsive to immune checkpoint inhibitor (ICB) therapy. Therefore, it is particularly important to identify effective and feasible combination treatment options for these patients. Methods: To construct an animal model of metastatic non-small cell lung cancer (A549 cells), the experimental group was treated with low dose radiotherapy (LDRT: The expression of local immune cells and their related molecules in the tumor microenvironment was evaluated at different time points after 1Gy) irradiation, and the effect and mechanism of LDRT and reasonable immunotherapy combination in the treatment of immune desert tumors were discussed. Follow-up clinical trials to evaluate the benefits and evidence of low-dose radiotherapy combined with immunotherapy induced advanced non-small cell lung cancer in humans. Results: 1. Low dose radiotherapy (LDRT) induced local immune cell infiltration in a mouse model of locally advanced in-situ non-small cell lung cancer 1Gy irradiation of tumors induces important transcriptional changes in vivo, particularly significant upregulation of inflammatory pathways, including IFN-α and IFN-γ responses, complement activation, IL6/JAK/STAT3 signaling pathways, and attracts the expression of key chemokines of T and NK cells, as well as cross-presented dendritic cells. Low-dose radiation therapy enhanced immunotherapy responsiveness in a mouse model of locally advanced in-situ non-small cell lung cancer Utilizing the pro-inflammatory effects of LDRT, the combination therapy showed a significant therapeutic effect on the initial low-T-cell inflammatory tumors. Low-dose radiation therapy combined with immunotherapy increases the number of local CD4 + and CD8 + T cells in the tumor. All components of the combination therapy help mobilize an effective anti-tumor immune response. Low dose radiotherapy combined with immune checkpoint blocking induced responses to advanced immune desert human tumors In patients with locally advanced non-small cell lung cancer treated with immunotherapy (IT) and low-dose radiotherapy (LDRT: 1Gy), low dose radiotherapy combined with immune checkpoint blocking has been clinically observed to induce responses to advanced immune desert human tumors. Conclusions: This paper demonstrates a novel and important synergy between LDRT and rationally developed combination immunotherapies for the treatment of tumors with poor immunoinfiltration that relies on simultaneous activation of multiple innate and adaptive immune pathways, revealing the interdependence between LDRT and immune regulation.

The dual role of chaperone-mediated autophagy in the response and resistance to cancer immunotherapy.

The dual role of chaperone-mediated autophagy in the response and resistance to cancer immunotherapy.