February 24, 2024

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Non-apoptotic regulatory cell death in tumor immunotherapy

Non-apoptotic regulatory cell death in tumor immunotherapy


Non-apoptotic regulatory cell death in tumor immunotherapy

In recent years, immunotherapy represented by immune checkpoint inhibitors ( ICIs ) has achieved unprecedented breakthroughs in cancer treatment.

However, many tumors respond poorly or even non-responsively to ICIs, in part due to a lack of tumor-infiltrating lymphocytes ( TILs ), which greatly limits the application of ICIs.

Transforming these immune “cold” tumors into “hot” tumors that may respond to ICIs is an unsolved problem in cancer immunotherapy.


Since anti-apoptosis is a pervasive feature of cancer, induction of non-apoptotic regulated cell death ( RCD ) is a novel cancer treatment strategy.

RCD plays a crucial role in maintaining homeostasis and disease development.

According to different morphological, biochemical, immunological and genetic features, RCD can be divided into two categories: apoptotic and non-apoptotic. Non-apoptotic RCD can be subdivided into autophagy , ferroptosis, pyroptosis, and necroptosis .

Recently, several studies have revealed the interaction between non-apoptotic RCD and antitumor immunity, and targeted therapy against autophagy, ferroptosis, pyroptosis and necroptosis combined with immunotherapy may exert powerful antitumor activity, Even tumors resistant to ICIs.

Therefore, it is necessary to gain an in-depth understanding of the multi-layered relationship between anti-tumor immunity and non-apoptotic RCD, as well as the potential targeted application of non-apoptotic RCD in improving the efficacy of immunotherapy in malignant tumors.





Autophagy is a regulatory mechanism that removes unnecessary or dysfunctional cellular components and recycles metabolic substrates.

In response to stress signals in the tumor microenvironment, autophagy pathways are altered in tumor cells and immune cells, resulting in differential effects on tumor progression, immunity, and therapy.


The tumor microenvironment ( TME ), plays a key role in cancer progression, metastasis and therapy resistance.

In the TME, autophagy in tumor cells can be induced by intracellular and extracellular stress signals, including metabolic stress, hypoxia, redox stress, and immune signals. ( For details, see Autophagy in Tumor Immunity and Therapy )


Evasion of antitumor immune responses is an important survival strategy for various tumors. Recent evidence suggests that autophagy plays an important role in tumor immune evasion.

It has been found that the downregulation of MHC class I molecules in pancreatic ductal adenocarcinoma ( PDAC ) is mediated through selective autophagic degradation, and inhibition of autophagy releases a strong antitumor immune response.


On the other hand, MDSCs play an immunosuppressive role in TME, and studies have shown that autophagy in MDSCs is a key mechanism for suppressing the antitumor immune activity of melanoma.

Autophagy in MDSC immune cells is central to the degradation of MHC class II molecules, preventing the initiation and activation of antitumor T cells.


Furthermore, autophagy, as a mechanism by which ( cancer ) cells respond to threatening stressors, is considered an important mechanism of therapy resistance in cancer therapy.

There is already evidence that the resistance of tumor cells to cisplatin is mediated, at least in part, by increased autophagy in ovarian cancer cell lines.

Similar evidence suggests that cisplatin, doxorubicin, and methotrexate overcome chemoresistance by inhibiting autophagy in osteosarcoma.


Autophagy inhibitors are divided into early-stage inhibitors against ULK1/ULK2 or VPS34, such as SBI-0206965, 3MA and wortmannin, and late-stage inhibitors against lysosomes, such as CQ, hydroxychloroquine (HCQ), bafilomycin A1 And monensin, CQ and HCQ inhibit autophagosome degradation by interfering with lysosomal acidification.

However, in clinical trials, HCQ monotherapy failed to control tumor growth in patients with advanced pancreatic cancer, and autophagy inhibition is currently combined with other cancer treatments to improve the therapeutic effect.




High autophagic flux in cancer is associated with reduced response to chemotherapy and with poor survival in cancer patients.

Preclinical studies have shown that inhibition of autophagy can overcome chemoresistance in NSCLC, bladder, thyroid, and pancreatic cancers.

In addition, results from some studies suggest that autophagy inhibition may synergize with inhibition of MEK–ERK signaling.


An early phase II study in 2014 used HCQ monotherapy in patients with metastatic pancreatic cancer who had previously been treated with other modalities, with a primary endpoint of two-month progression-free survival.

As a result, autophagy levels were reduced to varying degrees in different patients, but the primary endpoint was not significantly improved.

Another study combining HCQ, gemcitabine, and nab-paclitaxel in patients with advanced or metastatic pancreatic cancer also failed to demonstrate a 12-month extension of overall survival.

Importantly, however, patients on HCQ showed a significantly better response rate ( 38.2% vs. 21.1% ).



Autophagy plays a key role in protecting tumor cells from cell death induced by radiation therapy. In breast cancer cells, radiation induces the expression of autophagy-related genes, accompanied by accumulation of autophagosomes.

Short-term inhibition of autophagy at the same time as radiotherapy enhances the cytotoxicity of radiotherapy against drug-resistant cancer cells. Likewise, hypoxia enhanced the radioresistance of A549 lung cancer cells by inducing autophagy.


In glioblastoma, radiotherapy induces autophagy by increasing the expression of mammalian STE20-like protein kinase 4 ( MST4 ), which stimulates autophagy through ATG4B phosphorylation.

The small-molecule inhibitor NSC185058 ( targeting ATG4B ) in combination with radiotherapy impairs glioblastoma intracranial xenograft growth and prolongs survival in treated mice.

Therefore, targeting tumor autophagy may enhance the efficacy of radiotherapy. In fact, autophagy inhibitors combined with radiation therapy have been utilized in clinical trials in cancer patients.



Harnessing the immune system is an important way to fight cancer. Inhibition of autophagy may impair systemic immunity, as autophagy is involved in immune system development and effector T cell survival and function.

However, in preclinical models of melanoma and breast cancer, systemic inhibition of autophagy by CQ over a short period of time did not impair T cell function.

The data suggest that the immune system may be tolerant to some degree of autophagy inhibition.

However, given that autophagy can modulate tumor immune responses, targeting autophagy can improve the efficacy of immunotherapy and overcome immunotherapy resistance.


For example, inhibition of VPS34 kinase activity with inhibitors SB02024 or SAR405 resulted in increased levels of CCL5, CXCL10, and IFN-γ in the TME, resulting in increased levels of NK and T-cell tumor infiltration in melanoma and colorectal cancer models.

In these models, VPS34 inhibition also reversed resistance to anti-PD1 or anti-PD-L1 therapy. Furthermore, CQ treatment blocked autophagy-mediated MHC class I degradation and synergized with dual ICB treatment (anti-PD1 and anti-CTLA4 antibodies) to generate enhanced antitumor immune responses in a mouse model of pancreatic cancer.


Therefore, targeting autophagy may enhance immunotherapy. Clinical trials of HCQ combined with immunotherapy in patients with different types of cancer are currently underway.

Furthermore, autophagy regulation may offer some benefits to cancer patients treated with CAR-T cells. It is well known that the TME is a barrier to CAR-T cell infiltration and function in solid tumors , given that autophagy inhibition can remodel the TME and promote the production of TH1 -type chemokines, autophagy inhibition can promote CAR-T cell trafficking to tumors, CAR – Enhanced autophagy of T cells may support T cell fitness and survival in the TME .

In addition, tumor autophagy inhibition may lead to increased antigen expression, thereby enhancing CAR-T cell-mediated tumor killing. Finally, autophagy inhibition may ameliorate cytokine release syndrome and provide clinical benefit to patients.

Overall, the potential of inhibiting autophagy to enhance the efficacy of immunotherapy is a promising area that is constantly being explored.




Iron death


Ferroptosis is a recently discovered programmed cell death that plays an important role in tumor biology and therapy.

This unique form of cell death, characterized by iron-dependent lipid peroxidation, is precisely regulated by cellular metabolic networks including lipid, iron, and amino acid metabolism.

In addition, ferroptosis is also considered to be related to T cell-mediated antitumor immunity and affects the efficacy of tumor immunotherapy.


Ferroptosis is a regulated cell death caused by iron-dependent lipid peroxidation.

Three key features of ferroptosis have been deciphered: membrane lipid peroxidation, availability of intracellular iron, and loss of antioxidant defenses. ( For details, see ferroptosis in tumor immunotherapy )


Non-apoptotic regulatory cell death in tumor immunotherapy



It was recently found that ferroptosis contributes to the antitumor effect of CD8+ T cells and affects the efficacy of anti-PD-1/PD-L1 immunotherapy.

Immunotherapy combined with ferroptosis-promoting modalities, such as radiation therapy and targeted therapy, can have a synergistic effect through ferroptosis to promote tumor control.


Combination of immunotherapy and cystine restriction

Recently, it has been reported that CD8+ T cells activated by anti-PD-L1 immunotherapy promote tumor cell ferroptosis by secreting IFN-γ after PD-L1 blockade.

Secreted IFN-γ significantly downregulated the expression of SLC3A2 and SLC7A11 in tumor cells, resulting in decreased cystine uptake, enhanced lipid peroxidation, and subsequent ferroptosis.

Cystine/cysteinase synergizes with anti-PD-L1 to generate potent antitumor immunity by inducing ferroptosis.


Immunotherapy combined with targeted therapy

A recent study showed that resistance to anti-PD-L1 therapy can be overcome by combination with a TYR03 receptor tyrosine kinase (RTK) inhibitor, which promotes ferroptosis. Increased expression of TYR03 was found in anti-PD-1-resistant tumors.

Mechanistically, the TYR03 signaling pathway upregulates the expression of key ferroptosis genes such as SLC3A2, thereby inhibiting tumor-induced ferroptosis.

In a syngeneic mouse model of TNBC, inhibition of TYR03 promoted ferroptosis and sensitized tumors to anti-PD-1 therapy.

This study reveals that abolishing ferroptosis by using TYR03 inhibitors is an effective strategy to overcome immunotherapy resistance.


Immunotherapy combined with radiotherapy

Recent evidence suggests that the synergistic effect of radiotherapy and immunotherapy is associated with increased susceptibility to ferroptosis.

Radiation has been shown to induce ferroptosis, and genetic and biochemical signatures of ferroptosis have been observed in radiation-treated cancer cells.

The mechanism involves radiation-induced ROS generation and upregulation of ACSL4, resulting in enhanced lipid synthesis, increased lipid peroxidation, and subsequent membrane damage.

Therefore, the antitumor effect of radiotherapy can be attributed not only to DNA damage-induced cell death but also to the induction of ferroptosis.

Synergistic downregulation of SLC7A11 by radiotherapy and immunotherapy, mediated by the DNA damage-activated kinases ATM and IFN-γ, resulted in decreased cystine uptake, increased ferroptosis, and enhanced tumor control.

These studies reveal ferroptosis as a novel mechanism by which immunotherapy and radiation work synergistically.


Combined application of immunotherapy and T cell ferroptosis inhibitor

In addition to inducing neoplastic ferroptosis, T cells themselves may also undergo ferroptosis, which may attenuate their immune response. GPX4-deficient T cells rapidly accumulate membrane lipid peroxides and undergo ferroptosis.

Similar to cancer cells, ACSL4 is also essential for ferroptosis of CD8+ T cells and their immune function.


Recently, two studies have shown that CD36 expression is increased in CD8+ tumor-infiltrating lymphocytes.

T-cell-intrinsic CD36 promotes uptake of oxidized lipids and induces lipid peroxidation, leading to CD8+ T-cell dysfunction.

These findings reveal that CD8+ T cell ferroptosis is a novel mode of tumor immunosuppression and underscore the therapeutic potential of blocking CD36 to enhance anti-tumor immunity.

Notably, this study also suggests that GPX4 plays a role in regulating the antitumor function of CD8+ TILs.

Therefore, therapeutic induction of ferroptosis in cancer cells by GPX4 inhibitors may have unwanted on-target effects on T cells and produce undesirable toxicity.




Pyroptosis was first described in macrophages infected with S. typhurium and S. flexneri in the 1990s .

Although initially thought to be an apoptotic process, further studies have shown that this bacterial-induced cell death is critically dependent on caspase-1.

Pyroptotic cells share some features with apoptotic cells, such as chromatin condensation and DNA fragmentation, but can be distinguished by their intact nucleus, pore formation, cell swelling, and osmotic lysis.



Typically, pyroptotic cell rupture is achieved by pore-forming GSDM protein activation mediated by cysteine ​​proteases after binding damage-associated molecular patterns ( DAMPs ) or pathogen-associated molecular patterns ( PAMPs ).

These same cysteine ​​proteases may also directly or indirectly promote the maturation of pro-inflammatory cytokines that, together with DAMPs, initiate or sustain inflammatory responses when released.


Although the number of known pyroptotic pathways is likely to increase in the future, two main pathways and several alternative pathways have been elucidated.

Among the major pathways, pyroptosis is induced by GSDMD, involving inflammatory caspase-1 ( canonical pathway ) or caspase-4/5 ( non-canonical pathway ).

Among the alternative pathways, the one that has received the most attention is GSDME-induced pyroptosis via caspase-3. ( For details , see Tumor Immunity and Pyroptosis )


Non-apoptotic regulatory cell death in tumor immunotherapy



The ability of cell death to trigger an adaptive immune response is called immunogenic cell death ( ICD ).

Unlike apoptosis, which is essentially a process of immune tolerance, pyroptosis has a molecular mechanism that induces a strong inflammatory response and is considered a form of ICD in some cases.

Although the link between pyroptosis and anticancer immunity is unclear, an increasing number of studies suggest that pyroptosis-mediated tumor clearance is achieved by enhancing immune activation and function.


Non-apoptotic regulatory cell death in tumor immunotherapy



For example, methotrexate infused into cholangiocarcinoma ( CCA ) cells using tumor cell-derived microparticles ( TMPs ) to induce GSDME-mediated pyroptosis, which activates patient-derived macrophages and recruits neutrophils to Drug-directed tumor destruction at the tumor site.

Furthermore, when this methotrexate TMP delivery system was infused into the bile duct lumen of extrahepatic CCA patients, neutrophil activation and remission of biliary obstruction were observed in 25% of patients.


In addition, GSDME-mediated pyroptosis was also found to cause immune cell infiltration/activation in melanoma through a combination of BRAF and MEK inhibitors and lead to melanoma regression.

In another strategy, metformin, the most commonly used drug for the treatment of type 2 diabetes, can inhibit cancer cell proliferation by indirectly activating pyroptosis through caspase-3.


A series of small-molecule inhibitors targeting KRAS, EGFR, or ALK mutant lung cancers have also been found to induce pyroptosis through caspase-3-mediated GSDME cleavage after activation of the intrinsic mitochondrial apoptotic pathway.

In breast cancer cells, treatment with RIG-1 agonists triggers extrinsic apoptotic pathways and pyroptosis, activates STAT1 and NF-κB and upregulates lymphocyte recruitment of chemokines.

Thus, following RIG-1 activation in mice, a reduction in breast cancer metastasis and tumor growth was accompanied by an increase in tumor lymphocytes.


Another major obstacle faced by nearly all anticancer immunotherapy strategies is the dysregulation of the immunosuppressive tumor microenvironment.

To address this issue, Lu et al. engineered NK92 cells containing a chimeric costimulatory transforming receptor ( CCCR ), which converts inhibitory PD-1 signals into activating signals, effectively enhancing antitumor activity.

In vitro, CCCR-NK92 cells rapidly killed H1299 cells via GSDME-mediated pyroptosis and significantly inhibited tumor growth in vivo.


In addition, more and more exciting research reports show that pyroptosis induction synergizes with PD-1 inhibitors to turn tumors from “cold” to “hot”, indicating the great potential of this combination.






Necroptosis, proposed by Deggerev et al in 2005, is another form of ICD that includes specific death receptors ( DRs ) such as FAS and tumor necrosis factor receptor 1 ( TNFR1 ) or toll-like receptor 3 ( TLR3 ) ) and other PRRs recognize unfavorable signals from the intracellular and extracellular microenvironments, thereby triggering necroptosis.


Non-apoptotic regulatory cell death in tumor immunotherapy


There is evidence that necroptosis acts as a tumor suppressor in most cases.

In a study of more than 60 cancer cell lines, two-thirds of the samples showed reduced levels of RIPK3, suggesting that cancer cells tend to escape necroptosis to survive.

In addition, necroptosis is closely related to cancer prognosis, and RIPK3 expression is an independent prognostic factor for overall survival and disease-free survival in colorectal cancer patients.

Recently, a study showed that the expression of RIPK1, RIPK3 and MLKL was associated with better overall survival in patients with liver cancer.


Effectors in necroptosis such as RIPK1 and RIPK3 can directly regulate immune cell function independent of cell death.

It was found that RIPK3-mediated activation of phosphoglycerate mutase 5 ( PGAM5 ), through nuclear translocation of nuclear factor ( NFAT ) and dephosphorylation of dynamin-related protein 1 ( Drp1 ) in activated T cells , in an independent The necrotic pathway promotes natural killer T cell-mediated antitumor immune responses.

Furthermore, injection of RIPK3-activated necrotic tumor cells into pre-existing tumors enhanced antitumor immunity in syngeneic melanoma and lung adenocarcinoma models.


Necroptosis inducers can synergize with ICIs against tumors.

In melanoma, the SMAC analog Birinapant sensitizes tumor cells to TNF-α-mediated T cell killing and directly modulates immune cell function, including B cells, myeloid-derived cells, and cytotoxic lymphocytes, by modulating the NF-κB signaling pathway cells, thereby improving the response to ICIs.

Similarly, activation of CD8+ T cells and NK cells by SMAC analogs via RIPK1-dependent cell death enhanced the survival benefit of immune checkpoint blockade in mouse tumor models.

In addition, SMAC analogs are also believed to improve the efficacy of CAR-T cells in the treatment of acute lymphoblastic leukemia, since death receptor signaling is a key mediator of CAR-T cell cytotoxicity.





The study of non-apoptotic RCDs is a broad and rapidly developing field.

A new view is that targeting autophagy, pyroptosis, ferroptosis, and necroptosis in local tumors profoundly affects immune cells infiltrating the TME and the response to immunotherapy.

Extensive interactions exist between non-apoptotic cell death mechanisms and antitumor immunity.


Although the roles of autophagy, pyroptosis, ferroptosis, and necroptosis in tumor immunity remain unclear, several findings suggest more complex interactions between non-apoptotic RCDs and immunity in different tumor types and contexts , but targeting non-apoptotic cell death has increasingly emerged as a promising strategy to improve the efficacy of cancer immunotherapy.

Currently, it is imperative to develop more specific cell death-inducing drugs that act on tumor cells with minimal side effects on normal tissues.


At the same time, preclinical testing of these drugs in combination with ICIs, as well as chemotherapy, radiotherapy, and targeted therapy, may be critical in balancing treatment goals with possible adverse effects.

In the future, clinical trials of combined therapy should be actively encouraged to evaluate its efficacy and safety, and provide more references for subsequent in-depth research to benefit more cancer patients.







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2. Pyroptosis at the forefront of anticancerimmunity. J Exp Clin Cancer Res. 2021; 40: 264.

3. Ferroptosis: a promising target for cancer immunotherapy. Am J Cancer Res. 2021; 11(12): 5856–5863.

4. Autophagy in tumour immunity and therapy. Nat Rev Cancer. 2021 May; 21(5): 281–297.

5. Autophagy in Cancer Therapy-Molecular Mechanisms and Current Clinical Advances. Cancers (Basel). 2021 Nov8;13(21):5575.

Non-apoptotic regulatory cell death in tumor immunotherapy

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