Targeting TACC3 Induces Immunogenic Cell Death and Enhances T-DM1 Response in HER2-Positive Breast Cancer.

Trastuzumab emtansine (T-DM1) was the first and one of the most successful antibody-drug conjugates (ADC) approved for treating refractory HER2-positive breast cancer. Despite its initial clinical efficacy, resistance is unfortunately common, necessitating approaches to improve response. Here, we found that in sensitive cells, T-DM1 induced spindle assembly checkpoint (SAC)-dependent immunogenic cell death (ICD), an immune-priming form of cell death. The payload of T-DM1 mediated ICD by inducing eIF2α phosphorylation, surface exposure of calreticulin, ATP and HMGB1 release, and secretion of ICD-related cytokines, all of which were lost in resistance. Accordingly, ICD-related gene signatures in pretreatment samples correlated with clinical response to T-DM1-containing therapy, and increased infiltration of antitumor CD8+ T cells in posttreatment samples was correlated with better T-DM1 response. Transforming acidic coiled-coil containing 3 (TACC3) was overexpressed in T-DM1-resistant cells, and T-DM1 responsive patients had reduced TACC3 protein expression whereas nonresponders exhibited increased TACC3 expression during T-DM1 treatment. Notably, genetic or pharmacologic inhibition of TACC3 restored T-DM1-induced SAC activation and induction of ICD markers in vitro. Finally, TACC3 inhibition in vivo elicited ICD in a vaccination assay and potentiated the antitumor efficacy of T-DM1 by inducing dendritic cell maturation and enhancing intratumoral infiltration of cytotoxic T cells. Together, these results illustrate that ICD is a key mechanism of action of T-DM1 that is lost in resistance and that targeting TACC3 can restore T-DM1-mediated ICD and overcome resistance. SIGNIFICANCE: Loss of induction of immunogenic cell death in response to T-DM1 leads to resistance that can be overcome by targeting TACC3, providing an attractive strategy to improve the efficacy of T-DM1.

Multivalent Rhamnose-Modified EGFR-Targeting Nanobody Gains Enhanced Innate Fc Effector Immunity and Overcomes Cetuximab Resistance via Recruitment of Endogenous Antibodies.

Cetuximab resistance is a significant challenge in cancer treatment, requiring the development of novel therapeutic strategies. In this study, a series of multivalent rhamnose (Rha)-modified nanobody conjugates are synthesized and their antitumor activities and their potential to overcome cetuximab resistance are investigated. Structure-activity relationship studies reveal that the multivalent conjugate D5, bearing sixteen Rha haptens, elicits the most potent innate fragment crystallizable (Fc) effector immunity in vitro and exhibits an excellent in vivo pharmacokinetics by recruiting endogenous antibodies. Notably, it is found that the optimal conjugate D5 represents a novel entity capable of reversing cetuximab-resistance induced by serine protease (PRSS). Moreover, in a xenograft mouse model, conjugate D5 exhibits significantly improved antitumor efficacy compared to unmodified nanobodies and cetuximab. The findings suggest that Rha-Nanobody (Nb) conjugates hold promise as a novel therapeutic strategy for the treatment of cetuximab-resistant tumors by enhancing the innate Fc effector immunity and enhancing the recruitment of endogenous antibodies to promote cancer cell clearance by innate immune cells.

Regulation of pancreatic cancer therapy resistance by chemokines.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by high resistance and poor response to chemotherapy. In addition, the poorly immunogenic pancreatic tumors constitute an immunosuppressive tumor microenvironment (TME) that render immunotherapy-based approaches ineffective. Understanding the mechanisms of therapy resistance, identifying new targets, and developing effective strategies to overcome resistance can significantly impact the management of PDAC patients. Chemokines are small soluble factors that are significantly deregulated during PDAC pathogenesis, contributing to tumor growth, metastasis, immune cell trafficking, and therapy resistance. Thus far, different chemokine pathways have been explored as therapeutic targets in PDAC, with some promising results in recent clinical trials. Particularly, immunotherapies such as immune check point blockade therapies and CAR-T cell therapies have shown promising results when combined with chemokine targeted therapies. Considering the emerging pathological and clinical significance of chemokines in PDAC, we reviewed major chemokine-regulated pathways leading to therapy resistance and the ongoing endeavors to target chemokine signaling in PDAC. This review discusses the role of chemokines in regulating therapy resistance in PDAC and highlights the continuing efforts to target chemokine-regulated pathways to improve the efficacy of various treatment modalities.

GPRC5D-Directed CAR Yields 100% Response Rate.

Patients with refractory or relapsed multiple myeloma usually develop resistance to the two approved BCMA-targeting chimeric antigen receptor (CAR) T cells. A preliminary study of a new CAR T-cell therapy that zeroes in on the GPRC5D protein on multiple myeloma cells suggests that this approach is safe and effective. All 10 patients treated with the GPRC5D-targeting cells showed responses.

A combination of stromal PD-L1 and tumoral nuclear ß-catenin expression as an indicator of colorectal carcinoma progression and resistance to chemoradiotherapy in locally advanced rectal carcinoma.

Programmed cell death-1 (PD-1) and its ligand (PD-L1) are significant mediators of immune suppression in the tumor microenvironment. We focused on the immunological impact of PD-1/PD-L1 signaling during tumor progression in colorectal carcinoma (CRC) and its association with resistance to neoadjuvant chemoradiotherapy (NCRT) in locally advanced rectal carcinoma (LAd-RC). Histopathological and immunohistochemical analyses of 100 CRC cases (including 34 RC) without NCRT and 109 NCRT-treated LAd-RC cases were performed. Membranous tumoral PD-L1 expression was identified in 9 of 100 (9%) CRC cases, including 1 of 34 (2.9%) RC cases, but PD-L1 immunopositivity was not associated with any clinicopathological factors, with the exception of deficient mismatch repair (dMMR) status. In contrast, stromal PD-L1+ immune cells, which frequently exhibited coexpression of PD-1 and CD8 markers, were significantly correlated with tumor vessel invasion, nuclear ß-catenin+ tumor budding cancer stem cell (CSC)-like features, and unfavorable prognosis. In the LAd-RC cases, stromal CD8+ (but not PD-L1+) immune cell infiltration in pretreatment-biopsied samples was significantly and positively associated with therapeutic efficacy. After NCRT, tumoral PD-L1 expression was observed in only 2 of 83 (2.4%) tumors, independent of dMMR status, whereas high stromal PD-L1+ and tumoral nuclear ß-catenin positivity were significantly linked to a poor response to NCRT and high tumor budding features. In addition, high stromal PD-L1 immunoreactivity was significantly associated with poorer overall survival. In conclusion, a combination of stromal PD-L1+ immune cells and nuclear ß-catenin+ tumor budding may contribute to tumor progression in CRC and resistance to NCRT in LAd-RC, through formation of niche-like lesions that exhibit immune resistance and CSC properties.

Roles of HLA-G/KIR2DL4 in Breast Cancer Immune Microenvironment.

Human leukocyte antigen (HLA)-G is a nonclassical MHC Class I molecule, which was initially reported as a mediator of immune tolerance when expressed in extravillous trophoblast cells at the maternal-fetal interface. HLA-G is the only known ligand of killer cell immunoglobulin-like receptor 2DL4 (KIR2DL4), an atypical family molecule that is widely expressed on the surface of NK cells. Unlike other KIR receptors, KIR2DL4 contains both an arginine-tyrosine activation motif in its transmembrane region and an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail, suggesting that KIR2DL4 may function as an activating or inhibitory receptor. The immunosuppressive microenvironment exemplified by a rewired cytokine network and upregulated immune checkpoint proteins is a hallmark of advanced and therapy-refractory tumors. Accumulating evidence has shown that HLA-G is an immune checkpoint molecule with specific relevance in cancer immune escape, although the role of HLA-G/KIR2DL4 in antitumor immunity is still uncharacterized. Our previous study had shown that HLA-G was a pivotal mediator of breast cancer resistance to trastuzumab, and blockade of the HLA-G/KIR2DL4 interaction can resensitize breast cancer to trastuzumab treatment. In this review, we aim to summarize and discuss the role of HLA-G/KIR2DL4 in the immune microenvironment of breast cancer. A better understanding of HLA-G is beneficial to identifying novel biomarker(s) for breast cancer, which is important for precision diagnosis and prognostic assessment. In addition, it is also necessary to unravel the mechanisms underlying HLA-G/KIR2DL4 regulation of the immune microenvironment in breast cancer, hopefully providing a rationale for combined HLA-G and immune checkpoints targeting for the effective treatment of breast cancer.

Massive digital gene expression analysis reveals different predictive profiles for immune checkpoint inhibitor therapy between adenocarcinoma and squamous cell carcinoma of advanced lung cancer.

BACKGROUND: Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC. METHODS: This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch's t-test. RESULTS: Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma. CONCLUSIONS: This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma.

Overcoming resistance to immune checkpoint therapy in PTEN-null prostate cancer by intermittent anti-PI3Kα/ß/δ treatment.

Combining immune checkpoint therapy (ICT) and targeted therapy holds great promises for broad and long-lasting anti-cancer therapies. However, combining ICT with anti-PI3K inhibitors have been challenging because the multifaceted effects of PI3K on both cancer cells and immune cells within the tumor microenvironment. Here we find that intermittent but not daily dosing of a PI3Kα/ß/δ inhibitor, BAY1082439, on Pten-null prostate cancer models could overcome ICT resistance and unleash CD8+ T cell-dependent anti-tumor immunity in vivo. Mechanistically, BAY1082439 converts cancer cell-intrinsic immune-suppression to immune-stimulation by promoting IFNα/IFNγ pathway activation, ß2-microglubin expression and CXCL10/CCL5 secretion. With its preferential regulatory T cell inhibition activity, BAY1082439 promotes clonal expansion of tumor-associated CD8+ T cells, most likely via tertiary lymphoid structures. Once primed, tumors remain T cell-inflamed, become responsive to anti-PD-1 therapy and have durable therapeutic effect. Our data suggest that intermittent PI3K inhibition can alleviate Pten-null cancer cell-intrinsic immunosuppressive activity and turn "cold" tumors into T cell-inflamed ones, paving the way for successful ICT.

Naturally Occurring Telomerase-Specific CD4 T-Cell Immunity in Melanoma.

CD4 T cells play a key role in anticancer immunity. In this study, we investigate the clinical relevance of circulating CD4 T helper type 1 (Th1) response against telomerase (anti-TERT Th1 response) in patients with melanoma. The spontaneous anti-TERT Th1 response was detected in 54.5% (85/156) of patients with melanoma before treatment. The prevalence of this systemic response was inversely related to Breslow thickness >1 mm and American Joint Committee on Cancer stage ≥II (P = 0.001 and 0.032, respectively). In contrast to patients treated with targeted therapies, the anti-TERT Th1 immunity was associated with an objective response after immune checkpoint inhibitors treatment. Hence, 86% (18/21) of responder patients exhibited pre-existing anti-TERT Th1 versus 35% (6/19) in nonresponders (P = 0.001). This response was also associated with increased progression-free survival and overall survival in patients with melanoma treated with immune checkpoint inhibitors (P = 0.0008 and 0.012, respectively). Collectively, the presence of circulating anti-TERT Th1 response is inversely related to melanoma evolution and appears to be a predictive factor of response to immunotherapy. Our results highlight the interest in telomerase-specific CD4 Th1 response as a promising blood-based biomarker of immune checkpoint inhibitors therapy in melanoma.

A high OXPHOS CD8 T cell subset is predictive of immunotherapy resistance in melanoma patients.

Immune checkpoint inhibitor (ICI) therapy continues to revolutionize melanoma treatment, but only a subset of patients respond. Major efforts are underway to develop minimally invasive predictive assays of ICI response. Using single-cell transcriptomics, we discovered a unique CD8 T cell blood/tumor-shared subpopulation in melanoma patients with high levels of oxidative phosphorylation (OXPHOS), the ectonucleotidases CD38 and CD39, and both exhaustion and cytotoxicity markers. We called this population with high levels of OXPHOS "CD8+ TOXPHOS cells." We validated that higher levels of OXPHOS in tumor- and peripheral blood-derived CD8+ TOXPHOS cells correlated with ICI resistance in melanoma patients. We then developed an ICI therapy response predictive model using a transcriptomic profile of CD8+ TOXPHOS cells. This model is capable of discerning responders from nonresponders using either tumor or peripheral blood CD8 T cells with high accuracy in multiple validation cohorts. In sum, CD8+ TOXPHOS cells represent a critical immune population to assess ICI response with the potential to be a new target to improve outcomes in melanoma patients.

Construction of a novel immune-related lncRNA signature and its potential to predict the immune status of patients with hepatocellular carcinoma.

BACKGROUND: The accuracy of existing biomarkers for predicting the prognosis of hepatocellular carcinoma (HCC) is not satisfactory. It is necessary to explore biomarkers that can accurately predict the prognosis of HCC. METHODS: In this study, original transcriptome data were downloaded from The Cancer Genome Atlas (TCGA) database. Immune-related long noncoding ribonucleic acids (irlncRNAs) were identified by coexpression analysis, and differentially expressed irlncRNA (DEirlncRNA) pairs were distinguished by univariate analysis. In addition, the least absolute shrinkage and selection operator (LASSO) penalized regression was modified. Next, the cutoff point was determined based on the area under the curve (AUC) and Akaike information criterion (AIC) values of the 5-year receiver operating characteristic (ROC) curve to establish an optimal model for identifying high-risk and low-risk groups of HCC patients. The model was then reassessed in terms of clinicopathological features, survival rate, tumor-infiltrating immune cells, immunosuppressive markers, and chemotherapy efficacy. RESULTS: A total of 1009 pairs of DEirlncRNAs were recognized in this study, 30 of these pairs were included in the Cox regression model for subsequent analysis. After regrouping according to the cutoff point, we could more effectively identify factors such as aggressive clinicopathological features, poor survival outcomes, specific immune cell infiltration status of tumors, high expression level of immunosuppressive biomarkers, and low sensitivity to chemotherapy drugs in HCC patients. CONCLUSIONS: The nonspecific expression level signature involved with irlncRNAs shows promising clinical value in predicting the prognosis of HCC patients.

[Bispecific antibodies in onco-hematology: Applications and perspectives]. / Anticorps bispecifiques en oncohématologie : applications et perspectives.

Bispecific antibodies are novel approaches of immunotherapy engaging immune cells to destroy tumor cells. Their structure is variable and underlies their pharmacocinetic properties. These coumpounds are now being evaluated across multiple hematological malignancies. The anti-CD3/CD19 antibody blinatumomab is the first in class and have been approved for the treatment of patients with Ph-negative B-cell acute lymphoblastic leukemia. Other emerging applications are lymphoma, multiple myeloma and acute myeloid leukemia. The safety profile of bispecific antibodies is acceptable while limited by neurotoxicity and cytokine-release syndrome. The present review aims to depict the landscape of emerging bispecific antibodies currently in development for hematological malignancies.

[Bispecific antibodies in multiple myeloma]. / Myélome multiple et anticorps bispécifiques.

Immunotherapies have recently emerged as potential game changers in the treatment of multiple myeloma (MM). Those include monoclonal antibodies (targeting CD38 or CS1), bispecific antibodies (BsAb, mainly targeting BCMA, GPRC5D or FcRH5), antibody-drug conjugate (mainly targeting BCMA) and CAR-T cells (mainly targeting BCMA). BsAb have the capacity to bind two different antigens, one at the tumor cell surface and one on T cells (CD3), recreating the immune synapse. In this article, we discuss the main clinical data on BsAb in MM, as well as their different constructs and the potential mechanism of resistance.

Subclone-specific microenvironmental impact and drug response in refractory multiple myeloma revealed by single-cell transcriptomics.

Virtually all patients with multiple myeloma become unresponsive to treatment over time. Relapsed/refractory multiple myeloma (RRMM) is accompanied by the clonal evolution of myeloma cells with heterogeneous genomic aberrations and profound changes of the bone marrow microenvironment (BME). However, the molecular mechanisms that drive drug resistance remain elusive. Here, we analyze the heterogeneous tumor cell population and its complex interaction network with the BME of 20 RRMM patients by single cell RNA-sequencing before/after treatment. Subclones with chromosome 1q-gain express a specific transcriptomic signature and frequently expand during treatment. Furthermore, RRMM cells shape an immune suppressive BME by upregulation of inflammatory cytokines and close interaction with the myeloid compartment. It is characterized by the accumulation of PD1+ γδ T-cells and tumor-associated macrophages as well as the depletion of hematopoietic progenitors. Thus, our study resolves transcriptional features of subclones in RRMM and mechanisms of microenvironmental reprogramming with implications for clinical decision-making.

"γδT Cell-IL17A-Neutrophil" Axis Drives Immunosuppression and Confers Breast Cancer Resistance to High-Dose Anti-VEGFR2 Therapy.

Angiogenesis is an essential physiological process and hallmark of cancer. Currently, antiangiogenic therapy, mostly targeting the vascular endothelial growth factor (VEGF)/VEGFR2 signaling axis, is commonly used in the clinic for solid tumors. However, antiangiogenic therapies for breast cancer patients have produced limited survival benefits since cancer cells rapidly resistant to anti-VEGFR2 therapy. We applied the low-dose and high-dose VEGFR2 mAb or VEGFR2-tyrosine kinase inhibitor (TKI) agents in multiple breast cancer mouse models and found that low-dose VEGFR2 mAb or VEGFR2-TKI achieved good effects in controlling cancer progression, while high-dose treatment was not effective. To further investigate the mechanism involved in regulating the drug resistance, we found that high-dose anti-VEGFR2 treatment elicited IL17A expression in γδ T cells via VEGFR1-PI3K-AKT pathway activation and then promoted N2-like neutrophil polarization, thus inducing CD8+ T cell exhaustion to shape an immunosuppressive microenvironment. Combining anti-VEGFR2 therapy with immunotherapy such as IL17A, PD-1 or Ly-6G mAb therapy, which targeting the immunomodulatory axis of "γδT17 cells-N2 neutrophils" in vivo, showed promising therapeutic effects in breast cancer treatment. This study illustrates the potential mechanism of antiangiogenic therapy resistance in breast cancer and provides synergy treatment for cancer.

Immune-Related Genes to Construct a Novel Prognostic Model of Breast Cancer: A Chemosensitivity-Based Study.

Chemotherapy combined with surgery is effective for patients with breast cancer (BC). However, chemoresistance restricts the effectiveness of BC treatment. Immune microenvironmental changes are of pivotal importance for chemotherapy responses. Thus, we sought to construct and validate an immune prognostic model based on chemosensitivity status in BC. Here, immune-related and chemosensitivity-related genes were obtained from GSE25055. Then, univariate analysis was employed to identify prognostic-related gene pairs from the intersection of the two parts of the genes, and modified least absolute shrinkage and selection operator (LASSO) analysis was performed to build a prognostic model. Furthermore, we investigated the efficiency of this model from various perspectives, and further validation was performed using the Cancer Genome Atlas (TCGA) cohorts. We identified seven immune and chemosensitivity-related gene pairs and incorporated them into the Cox regression model. After multilevel validation, the risk model was found to be closely related to the survival rate, various clinical characteristics, tumor mutation burden (TMB) score, immune checkpoints, and response to chemotherapeutic drugs. In addition, the model was verified to exhibit predictive capacity as an independent factor over other candidate clinical features. Notably, the constructed nomogram was more accurate than any single factor. Altogether, the risk score model and the nomogram have potential predictive value and may have important practical implications.

Mechanisms of primary and acquired resistance to PD-1/PD-L1 blockade and the emerging role of gut microbiome

As a very promising immunotherapy, PD-1/PD-L1 blockade has revolutionized the treatment of a variety of tumor types, resulting in significant clinical efficacy and lasting responses. However, these therapies do not work for a large proportion of patients initially, which is called primary resistance. And more frustrating is that most patients eventually develop acquired resistance after an initial response to PD-1/PD-L1 blockade. The mechanisms that lead to primary and acquired resistance to PD-1/PD-L1 inhibition have remained largely unclear. Recently, the gut microbiome has emerged as a potential regulator for PD-1/PD-L1 blockade. This review elaborates on the current understanding of the mechanisms in terms of PD-1 related signaling pathways and necessary factors. Moreover, this review discusses new strategies to increase the efficacy of immunotherapy from the perspectives of immune markers and gut microbiome (AU)

A nanounit strategy reverses immune suppression of exosomal PD-L1 and is associated with enhanced ferroptosis.

In addition to increasing the expression of programmed death-ligand 1 (PD-L1), tumor cells can also secrete exosomal PD-L1 to suppress T cell activity. Emerging evidence has revealed that exosomal PD-L1 resists immune checkpoint blockade, and may contribute to resistance to therapy. In this scenario, suppressing the secretion of tumor-derived exosomes may aid therapy. Here, we develop an assembly of exosome inhibitor (GW4869) and ferroptosis inducer (Fe3+) via amphiphilic hyaluronic acid. Cooperation between the two active components in the constructed nanounit induces an anti-tumor immunoresponse to B16F10 melanoma cells and stimulates cytotoxic T lymphocytes and immunological memory. The nanounit enhances the response to PD-L1 checkpoint blockade and may represent a therapeutic strategy for enhancing the response to this therapy.

Environmental eustress modulates ß-ARs/CCL2 axis to induce anti-tumor immunity and sensitize immunotherapy against liver cancer in mice.

Although psycho-social stress is a well-known factor that contributes to the development of cancer, it remains largely unclear whether and how environmental eustress influences malignant diseases and regulates cancer-related therapeutic responses. Using an established eustress model, we demonstrate that mice living in an enriched environment (EE) are protected from carcinogen-induced liver neoplasia and transplantable syngeneic liver tumors, owning to a CD8+ T cell-dependent tumor control. We identify a peripheral Neuro-Endocrine-Immune pathway in eustress, including Sympathetic nervous system (SNS)/ß-adrenergic receptors (ß-ARs)/CCL2 that relieves tumor immunosuppression and overcomes PD-L1 resistance to immunotherapy. Notably, EE activates peripheral SNS and ß-ARs signaling in tumor cells and tumor infiltrated myeloid cells, leading to suppression of CCL2 expression and activation of anti-tumor immunity. Either blockade of CCL2/CCR2 or ß-AR signaling in EE mice lose the tumor protection capability. Our study reveales that environmental eustress via EE stimulates anti-tumor immunity, resulting in more efficient tumor control and a better outcome of immunotherapy.

PI3Kγδ inhibitor plus radiation enhances the antitumour immune effect of PD-1 blockade in syngenic murine breast cancer and humanised patient-derived xenograft model.

INTRODUCTION: We hypothesised that the combined use of radiation therapy and a phosphoinositide 3-kinaseγδ inhibitor to reduce immune suppression would enhance the efficacy of an immune checkpoint inhibitor. METHODS: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumours were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a phosphoinositide 3-kinase (PI3K)γδ inhibitor were then administered every other day for 2 weeks. The same experiments were performed in humanised patient-derived breast cancer xenograft model and its tumour was sequenced to identify immune-related pathways and profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. RESULTS: Using a PI3Kγδ inhibitor, radiation therapy (RT) and PD-1 blockade significantly delayed primary tumour growth, boosted the abscopal effect and improved animal survival. RT significantly increased CD8+cytotoxic T-cell fractions, immune-suppressive regulatory T cells (Tregs), myeloid-derived suppressor cells and M2 tumour-associated macrophages (TAMs). However, the PI3Kγδ inhibitor significantly lowered the proportions of Tregs, myeloid-derived suppressor cells and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumour CD8+ T-cell populations after triple combination therapy. In a humanised patient-derived breast cancer xenograft model, triple combination therapy significantly delayed tumour growth and decreased immune-suppressive pathways. In The Cancer Genome Atlas cohort, high Treg/CD8+ T cell and M2/M1 TAM ratios were associated with poor overall patient survival. CONCLUSION: These findings indicate PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME, and combining PI3Kγδ inhibitor, RT and PD-1 blockade may overcome the therapeutic resistance of immunologically cold tumours. SYNOPSIS: Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may be a viable clinical approach, helping to overcome the therapeutic resistance of immunologically cold tumours such as breast cancer.