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In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. 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. Once primed, tumors remain T cell-inflamed, become responsive to anti-PD-1 therapy and have durable therapeutic effect.
Immune checkpoint therapies ICT , such as those mediated by anti-PD-1 or CTLA-4 antibodies, have shown promising long-lasting effects on certain cancer types by activating T cell-mediated anti-tumor immunity 1.
Although the mechanisms underlying cancer-mediated T cell exclusion are largely unknown, it has become clear that promoting T cell infiltration may increase the range of cancers sensitive to ICT 5 , 6. Prostate cancer is the most common malignancy in males, and the second leading cause of male cancer-related death in the Western world 7.
Androgen deprivation therapy ADT is the mainstream treatment for prostate cancer. However, despite initial regression, many patients progress to highly aggressive castration-resistant prostate cancer CRPC , a disease stage with limited treatment options 8. Recent ICT trials on CRPC patients have shown disappointing results 9 , 10 , 11 , most likely due to low mutational load and defects in T cell-mediated anti-tumor immunity.
Therefore, treatments that can promote T cell infiltration may pave the way for efficient ICT on prostate cancers. Cancer cell-intrinsic PI3K activation in the Pten -null model also promotes an immune suppressive microenvironment, including increased immune suppressive myeloid-derived suppressor cells MDSC and regulatory T cells Treg , decreased dendritic cell maturation, as well as decreased T cell infiltration and activation 18 , 19 , 20 , These results suggest that inhibiting cancer cell-intrinsic immune suppressive activity induced by PI3K activation may be a prerequisite for promoting T cell infiltration and achieving anti-tumor immunity.
As one of the most important oncogenic pathways, PI3K activation promotes cell proliferation, survival, migration, angiogenesis, metabolic reprograming as well as an immune suppressive environment 22 , 23 , 24 , 25 ; on the other hand, PI3K is also a critical regulator for the functions of immune cells within the tumor microenvironment, and inhibition of PI3K activity in these immune cells may be detrimental for ICT 26 , 27 , Various immune cells within the tumor microenvironment are preferentially relying on different isoforms of the PI3K to promote or inhibit tumor development 24 , We confirmed this clinical observation on the Pten -null prostate cancer model.
However, most of the T cells remained in the stroma area and could not penetrate into the tumor acini even after castration Supplementary Fig. We then sought to test if T cells-induced upon castration could lead to anti-tumor immunity in the presence of ICT. Castrated Pten -null mice were treated with either the anti-PD-1 or isotype control antibody for 4 weeks Supplementary Fig. Therefore, the Pten -null prostate cancer model mimics human prostate cancers with poor T cell infiltration and resistance to anti-PD-1 monotherapy.
Pten -null prostate cancer model could serve as an in vivo model to investigate the molecular mechanism underlying the T cell exclusion phenotype-associated with human prostate cancers. We have shown in our previous study that PTEN-loss in the prostatic epithelial cells promotes immunosuppressive microenvironment during prostate cancer initiation and progression but the underlying mechanisms are largely unknown We hypothesized that PTEN-loss or PI3K pathway activation in cancer cells may modulate anti-cancer immune response, which dampens the immune activities of cells within the tumor microenvironment.
As expected for PI3K pathway inhibition, global gene expression analysis demonstrated that BAY treatment led to downregulation of mTOR signaling and cell proliferation-related pathways as well as Ki67 expressions in all 4 prostate cancer cell lines Fig. Statistical test was performed by GSEA. Culture supernatant was collected and analyzed as suggested by manufactural recommendation.
The experiment was repeated 3 independent times with similar results. Statistical test done by two-sided T test. Source data and exact p values are provided in the Source Data file. Importantly, many genes within this core were known to cause resistance to anti-PD-1 therapy when deleted 32 , and substantially overlapped with those recently identified with cancer-intrinsic evasion of killing by T cell function The potent effect of BAY in inhibiting cancer cell-intrinsic immunosuppressive activity prompted us to test whether BAY treatment could turn Pten -null prostate cancer to T cell-inflamed and promote T cell-mediated anti-tumor immunity in vivo.
A A schematic illustration of treatment schedules. B FACS analyses for tumor-associated immune cells. Prostates were fixed and stained with HE, and cancer cell areas in anterior lobes were measured and fold of differences between vehicle and treatment groups were presented.
Dashed red lines: the boundaries between cancer acini and stroma areas. We therefore searched for alternative dosing schedule, including intermittent and weekly treatment, to effectively target cancer cells while reducing immune toxicity effect We and others have shown the important roles of MDSC in establishing immunosuppressive microenvironment in the Pten null model 18 , 20 , 21 , Thus, intermittent but not daily BAY treatment could convert the immunosuppressive tumor microenvironment-associated with the Pten -null prostate cancer to a T cell-inflamed one.
This experiment was repeated 3 times the percentages of cell growth as compared to vehicle controls were presented. IC50s were calculated by GraphPad Prism 6 software. We then quantified tumor-associated Tregs in the Pten -null model in vivo.
The relative expression levels of Il2 , Cd25 and Cd40l were presented. Pten -null; Cd8- KO mice developed prostate cancer with similar characteristics as the Pten -null mice Fig. We also treated castrated Pten -null mice with a CD8 depletion antibody. Data were presented as dot plots with mean as the central lines; N.
We next tested whether the BAY induced T cell-inflamed phenotype persists without continuous drug administration for subsequent combination of ICT. Castrated Pten -null mice were treated with vehicle or BAY-I for 4 weeks, then the treatment was stopped for 4 or 10 weeks before the analyses. Interestingly, tumor size and weight were decreased significantly in both 4- and week drug withdrawal groups, as compared to the vehicle or BAY-I group after the last dose Fig.
Red dash line marks the boundary between cancer acini and stroma areas. D RNA-seq analysis shows T cell inflamed phenotype after drug withdrawal. The relative expression levels of indicated genes in each sample were determined and the statistical analysis was performed based on the average of expression levels of each cohort.
RNA-seq analysis of the bulk tumor tissues revealed that BAY-I treatment could increase the T cell-inflamed gene expression profile 12 and the expressions of MHC class I and II molecules, which were well maintained in 4-week drug withdrawal group Fig.
Importantly, the dendritic cell DC -associated genes were significantly upregulated in the drug withdrawal group Fig. Together, these results demonstrated that BAY-I treatment can prime the tumor and generate a persistent T cell inflammatory environment even in the absence of continued drug administration. Effective priming of tumor antigen-specific T cells requires secondary lymphoid organs such as lymph nodes 6. In Pten -null prostate tumor tissues, we found tertiary lymphoid structures TLS with clear B and T cell zones, resembling germinal center morphology 45 Fig.
Castrated Pten -null mice were treated with 4 cycles of BAY-I then drug was withdrawal for 4—10 weeks. The same staining were performed with 3 mice and similar results were observed. Castrated Pten -null mice were treated with 4 cycles of vehicle, BAY-I then withdrawal for 4 weeks, or treated with anti-PD-1 antibody as indicated in Fig.
Correlation between TLS score and Cd8a expression was calculated. Statistic was performed by Pearson correlation coefficient.
The TLS may also account for persistent T cell-inflamed phenotype after drug withdrawal. On the other hand, late exhaustion marker TIM-3 and CTLA-4 were not expressed on the cell surface even through their gene expressions were upregulated Figs.
A schematic illustration of treatment strategy. The same staining were performed with 6 mice in all cohort and similar results were observed. Data were presented as mean with dot plots; N. To test this, we first treated castrated Pten -null prostate cancer model with BAY-I for 4 weeks, then dosed with control or PD-1 antibody for 4 weeks Fig.
The mouse body weights did not change significantly Supplementary Fig. Sequential BAY-I and anti-PD-1 combination treatment significantly decreased cancer cell areas than isotype treated cohort, while this combination effect was absent, in the Pten -null; Cd8- KO prostate Fig.
As successful immunotherapy often associated with long-lasting durable therapeutic effects 50 , we investigated the potential long-term effect of sequential combination of BAY-I and anti-PD-1 therapy. Together, these results indicated that a sequential combination of BAY-I and anti-PD-1 therapy could lead to a long-lasting, durable immune cell-mediated anti-tumor effect even after complete drug withdrawal. We demonstrate in this study that a carefully designed isoform-specificity and dosing schedule for PI3K inhibitor, and sequential administration of targeted and anti-PDmediated ICT can effectively overcome resistance to ICT in a preclinical prostate cancer setting and achieve a long-last durable immune cell-mediated therapeutic effect even after drug withdrawal.
These are cancer cell intrinsic property and are reversible upon BAY withdrawal 30 Fig. Our study showed that not only the isoform profile but also the dosing schedule of BAY is critically important for promoting anti-cancer immunity. The differential effects of daily vs. Intermittent treatment could also minimize aberrant immune activation in non-cancerous organs, avoiding adverse side-effects Supplementary Fig.
Intermittent dosing of PI3K inhibitors have been reported by other works and shown to reach successful therapeutic effect while improve drug tolerance 35 , 53 , Optimization the dosing schedule of these inhibitors may also improve their therapeutic effects as monotherapies or in combination with ICT.
A recent report demonstrates a cloning replacement of tumor-specific T cells following ICT in human basal or squamous cell carcinoma, and suggest that pre-existing tumor-specific T cells may have limited role in ICT The different conclusions on the origins of tumor-specific T cells in our study and those by Yost et al. However, other immune cells may also contribute to the overall therapeutic outcome.
Together, the superior effects of intermittent BAY treatment support the idea that drugs that can co-target both cancer cell-intrinsic and microenvironment pathways may have considerably more clinical benefit than single-target drugs.
Treatments that can improve T cell infiltration may augment ICT efficacy 2 , 5 , Intriguingly, once the tumor has become T cell-inflamed, it stays in T cell-inflamed status even after drug withdrawal Figs.
This T cell-inflamed state paves the way for successful anti-PD1 treatment. Importantly, the T cell-inflamed state and memory T cell signature remain even after 4 weeks of anti-PD1 antibody withdrawal, demonstrating long-last and durable anti-tumor immunity Fig.
Although the detailed mechanisms associated with this prolonged response require further investigation, our study provides a successful pre-clinical case for sequential, instead of simultaneous, anti-PI3K and ICT combination treatment to avoid potential combined toxicity when both drugs are used together.
Future works are need to test the effects of BAY-I and sequential BAY-I and anti-PD-1 treatment strategies on other models with different cancer-initiating and immune evasion mechanisms, and in humanized mouse cancer models before moving to clinical settings.
In summary, our results demonstrate that a carefully designed anti-PI3K treatment, both in its specificity and dosing schedule, to inhibit cancer cell growth while promote anti-tumor immunity, is critically important for successful combination of anti-PI3K targeted therapy and ICT.
For surgical castration, mouse was firstly anaesthetized by avertin. Then, a midline ventral skin incision was made into the scrotum and testicles were removed from both sides by sealing off the blood vessels using suture line Jinhuan Medical, CR The skin incision was then sutured using suture line. The animals were closely monitored during the procedure for signs of pain or bleeding and placed in a clean cage for post-surgery recovery.
The signal was detected by using ECL western blotting substrate kit Thermo fisher, and The following secondary antibodies were used: HRP-conjugated anti-mouse antibody Jackson ImmunoResearch Laboratories, ; Image Lab 5. Prostates were dissected and photographed, weighted and minced in sterile tissue culture dishes, and subjected to collagenase A 1. Data was analyzed by using flowjo software.
Cells were seeded at a density of 2. Four days later, cells were harvested, live cell numbers were counted by cell counter, and T cell proliferation rate proliferated cell was determined by CFSE staining was determined by FACS. Cell growth rate was calculated by the percentage of live, proliferated cell number at each drug concentration vs.
BAY was dissolved in 0. The slide was washed in PBS pH 7. The antibodies used and dilutions were listed in Supplementary Table 2. All antibodies used are listed in Supplementary Table 2. Primers were listed in Supplementary Table 2. All RNA-seq data were aligned to the mm10 genome using Tophat version v2. Differentially expressed genes were identified by Cuffdiff version v2. FPKM was used for following analysis and comparison. GSEA analysis was performed as software suggested The pathway activity score was calculated with GSVA The expression levels of TLS score genes 46 were scaled to the range of 0 to 1, and TLS score was defined as the mean scaled value of related genes for each individual sample,.
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