CD: Chinese scientists break the predicament of liver cancer immunotherapy resistance!

Globally, the incidence of liver tumors (hepatocellular carcinoma, HCC) continues to increase, becoming an important factor endangering public health [1]
.

It is predicted that by 2025, there will be more than one million new HCC patients every year [2]
.

According to the data released by the International Agency for Research on Cancer of the World Health Organization, the incidence of HCC in my country ranks eighth in the world in 2020, and among all cancers in China, the incidence of HCC ranks fifth and the mortality rate ranks second.
Prevention and treatment are urgently needed! In the past five years, immune checkpoint inhibitors (ICIs) have revolutionized HCC treatment
.

Compared with the traditional first-line treatment of HCC, sorafenib, various combination therapy options, such as the combination therapy of atezolizumab (PD-L1) and bevacizumab (VEGF-A), duvalizumab Combination therapy of Utuzumab (PD-L1) and Tremelimumab (CTLA-4), combination therapy of atezolizumab and Cabozantinib (multi-target tyrosinase inhibitor), etc.
, have achieved better results.
therapeutic effect
.

In addition, the combination therapy of nivolumab (PD-1) and ipilimumab (CTLA-4), and the monotherapy of pembrolizumab (PD-1) are especially excellent The early clinical data of the US Food and Drug Administration (FDA) entered the fast-track approval channel [3]
.

However, the objective response rate (ORR) of HCC patients in Asia to ICI regimens is not ideal [4], therefore, it is extremely important to understand the immunosuppressive microenvironment of HCC, how to break immunotherapy tolerance, and develop novel immunotherapies The plan became the top priority
.

Recently, Academician Fan Jia from Zhongshan Hospital Affiliated to Fudan University, Zhu Di from the Department of Pharmacology, School of Basic Medicine, Fudan University, and Xu Yang's team from Zhongshan Hospital Affiliated to Fudan University collaborated to publish important research results in the famous journal Cancer Discovery
.

Using clinical studies of HCC patients and mouse HCC models, they found that interferon (IFN)-α inhibits the glycolysis of HCC cells by regulating the transcriptional activity of FosB in HCC cells, and inhibits the hypoxia-inducible factor HIF1α, thereby reducing the response to glucose.
Uptake, remodel and form a glucose-rich tumor microenvironment (TME); high-glucose TME promotes the activation of the mTOR-FOXM1 signaling pathway in T cells, and makes tumor-infiltrating CD8+ T cells highly express the costimulatory molecule CD27, enhancing their killing function.
Amplify the therapeutic effect of PD-1 blockade on HCC[5]
.

More importantly, the IFN-α+PD-1 blockade combination therapy regimen had a high response rate in HCC patients and significantly inhibited the disease progression of HCC
.

 Their research results provide a solid and reliable theoretical basis for the development of new immunotherapy options for HCC, and the results of their pre-clinical trials are very exciting! The attending physician Hu Bo of Zhongshan Hospital Affiliated to Fudan University, Dr.
Yu Mincheng of Zhongshan Hospital, Dr.
Ma Xiaolu of Zhongshan Hospital, Dr.
Sun Jialei of Zhongshan Hospital, and Dr.
Liu Chenglong of School of Pharmacy are the co-first authors of this article
.

Next, let's take a look at how the team of Academician Fan Jia/Zhu Di/Xu Yang explored the therapeutic effect of IFN-α+PD-1 blockade on HCC and the immunological mechanism behind it
.

IFN-α+PD-1 blockade combined therapy significantly inhibited tumor development.
To observe the combined treatment effect of IFN-α+PD-1 blockade, researchers observed 15 patients with liver cancer that could not be surgically removed, and were given IFN-α and PD-1 blockade.
α+PD-1 blockade combination therapy
.

Surprisingly, the ORR was 40.
0% and the disease control rate (DCR) was as high as 80.
0% after the combination therapy, while there were no treatment-related deaths
.

Patient No.
1 was a 67-year-old woman who had undergone arterial chemoembolization (TACE) and subsequently received PD-1 blockade, but the clinical response was small, resulting in persistent disease progression
.

The largest tumor (2.
3 × 4.
1 cm) in this patient's liver was located in segment VIII, near the branch of the hepatic portal vein
.

However, after 7 weeks of combined treatment with IFN-α + PD-1 blockade, the patient's tumor developed necrosis and its size was significantly smaller
.

At later follow-up observations, the patient's tumor size did not increase, nor did he experience side effects
.

Patient 2 was a 34-year-old man with a very large liver cancer
.

The patient underwent multiple TACE treatments and two liver resections, followed by PD-1 blockade as well
.

The patient had no obvious signs of liver cancer recurrence within one year of treatment
.

But two years later, the patient developed symptoms of dyspnea, and a CT test found that there were more than 10 tumor metastases in the lungs
.

Fortunately, after two months of IFN-α + PD-1 blockade combination therapy, CT examination found that multiple tumor metastases in the patient's lungs were completely removed, and the largest tumor nodule (1.
4 cm) was found.
Significant shrinkage and no new metastases appeared
.

IFN-α+PD-1 blockade combination therapy promotes the ratio of CD27+CD8+ T cells Cell construction of an in situ syngeneic mouse liver cancer model
.

The results showed that compared with monotherapy such as IFN-α or PD-1 blockade, the combined treatment of IFN-α+PD-1 blockade could significantly inhibit the occurrence and development of tumors, and at the same time, the survival time of tumor-bearing mice was significantly prolonged
.

More importantly, IFN-α+PD-1 blockade combination therapy can induce tumor necrosis, thereby completely clearing the metastases in the lung
.

Further analysis found that IFN-α+PD-1 blockade combination therapy relies on a healthy immune system, while IFN-α directly acts on HCC cells expressing its receptor (IFNAR1)
.

To understand the effect of IFN-α+PD-1 blockade combination therapy on immune cells infiltrating the TME, we isolated CD45+ immune cells from tumor tissue and used mass cytometry (CyTOF) to detect 42 cell surface or intracellular immune markers
.

It was found that IFN-α+PD-1 blockade combination therapy significantly reduced the number of depleted CD8+ T cell precursors
.

Next, the researchers performed depletion experiments of CD4+ T cells and CD8+ T cells in Hepa1-6 tumor-bearing mice
.

It was found that CD8+ T cells played an important role in it
.

Combining the detection results of CyTOF and multicolor immunofluorescence, the researchers observed a significant increase in the number of CD27+CD8+ T cell subsets in tumor tissue after combined treatment with IFN-α+PD-1 blockade
.

In addition, the researchers co-cultured HCC patient-grown organoids (PDOs) with autologous tumor-infiltrating immune cells and observed a similar phenomenon, that is, the combined treatment of IFN-α + PD-1 blockade can maximize the increased CD27 expression on CD8+ T cells
.

So, can CD27 expression promote the anti-tumor function of CD8+ T cells? To this end, the researchers used Cd27-specific siRNA to knock down the expression of CD27 on tumor-infiltrating CD8+ T cells
.

As expected, the ability of CD8+ T cells to secrete inflammatory cytokines and cytotoxic granules was significantly reduced after CD27 decline
.

At the same time, the single-cell sequencing results of tumor tissues from HCC patients also confirmed that the expression levels of cytotoxicity and proliferation-related genes on CD27highCD8+ T cells were also significantly increased
.

Interestingly, the expression of PD-1 on CD27highCD8+ T cells in tumor tissues of HCC patients was also higher than that on CD27lowCD8+ T cells
.

The above results suggest that CD27+CD8+ T cell subsets may play an important role in the process of IFN-α+PD-1 blockade combination therapy
.

IFN-α+PD-1 blockade combination therapy inhibited tumor cell glycolysis gene set enrichment analysis (GSEA) and Western blot experiments confirmed that blocking PD-1 would promote HIF1α signaling pathway in tumor cells, however, combined Treatment significantly inhibited the HIF1α signaling pathway
.

In contrast to tumor cells, the expression of HIF1α and its downstream molecules decreased in tumor-infiltrating CD8+ T cells after PD-1 blockade, while the combined treatment of IFN-α+PD-1 blockade had the opposite effect.
As a result, the ability of CD8+ T cells to take up glucose is enhanced, thereby promoting their proliferation and activation
.

IFN-α negatively regulates HIF1α through the IRF1-FosB signaling axis.
HIF1α plays an important role in glycolysis, so we performed extracellular acidification rate (ECAR) experiments
.

Consistent with the changes in HIF1α signaling pathway, blocking PD-1 can promote glycolysis in tumor cells, while the combined treatment of IFN-α + PD-1 blockade significantly inhibits glycolysis in tumor cells
.

Contrasting results were obtained in CD8+ T cells
.

Furthermore, after blocking PD-1, glucose levels in the TME were significantly reduced, whereas IFN-α treatment reversed this change
.

IFN-α+PD-1 blockade combined therapy activates the mTOR-FOXM1 signaling pathway.
In-depth studies have found that IFN-α treatment can promote the expression of the transcription factor IRF1 in tumor cells and cause it to accumulate in the nucleus, thereby inhibiting the expression of the tumor-promoting gene FosB.
expression, and FosB will bind to HIF1α, resulting in HIF1α unable to continue to promote the expression of glycolysis-related genes, such as Pfkl, Hk1, Pkm2 and Ldha, etc.
, and finally cause the tumor cell glycolysis capacity to decline, forming a TME with high glucose content
.

The higher the glucose content in the TME, the higher the protein levels of molecules such as p-mTOR, HIF1α, PKM2, LDHA and CD27 in CD8+ T cells, and this process is dependent on the mTOR signaling pathway
.

To investigate the regulatory role of mTOR on CD27, the researchers used the Signaling Pathway Project (SPP) database to predict transcription factors that regulate CD27 in human leukocytes and integrated with single-cell data
.

It was found that CD27highCD8+ T cells highly expressed FOXM1
.

Although at steady state, overexpression of Foxm1 in CD8+ T cells could not increase the proportion of CD27+CD8+ T cells, but significantly promoted the secretion of IFN-γ and IL-2 from CD8+ T cells
.

Interestingly, the expression of FOXM1 in CD8+ T cells was significantly increased after IFN-α treatment, which was further amplified by the combined treatment of IFN-α+PD-1 blockade
.

In addition, high glucose medium also promoted FOXM1 expression
.

In contrast, mTOR inhibitors or HIF1α inhibitors reversed FOXM1 expression
.

Subsequently, the researchers treated tumor-bearing mice with IFN-α+PD-1 blockade combination, and isolated tumor-infiltrating CD8+ T cells.
Both overexpression and knockdown experiments confirmed that FOXM1 can promote CD8+ T cells to express CD27
.

In addition to this, the researchers also saw a similar phenomenon in the TCGA database and single-cell sequencing data of HCC, that is, the expression levels of CD27 and FOXM1 were significantly positively correlated
.

CD27 and CD8 are important indicators of good prognosis in HCC patients.
The above results confirm that IFN-α+PD-1 blockade combined therapy can remodel the TME, increase the glucose content in the microenvironment, activate the mTOR signaling pathway in CD8+ T cells, and increase the FOXM1 expression levels, thereby enhancing Cd27 transcription
.

Finally, the researchers analyzed the survival prognosis of HCC patients, and found that the more CD27highCD8high cells, the longer the patients' progression-free survival and overall survival, and the better the prognosis
.

Not only that, after PD-1 blockade treatment in HCC patients, more tumor-infiltrating CD27+CD8+ lymphocytes were found in responding patients
.

Overall, the research team of Academician Fan Jia/Zhu Di/Xu Yang found that IFN-α+PD-1 blockade combination therapy can significantly inhibit tumor growth in liver cancer mice or HCC patients
.

In-depth studies have confirmed that IFN-α can induce the expression of IRF1 in HCC cells, and then inhibit the transcription of FosB, resulting in the inhibition of the transcriptional activity of HIF1α, which reduces the glycolytic capacity of HCC cells and remodels the TME
.

Glucose-rich TME in turn activates the mTOR-FOXM1 signaling pathway of CD8+ T cells, resulting in increased expression of the costimulatory molecule CD27 and promoting the tumor immune surveillance function of CD8+ T cells
.

Summary of article content Academician Tu Fanjia and his team have a long way to go.
The IFN-α+PD-1 blockade combination therapy they designed is still immature.
For example, the clinical patient population of HCC should be expanded to test IFN-α+ The clinical value of PD-1 blockade combination therapy; the adverse reactions of this treatment plan need to be systematically evaluated; at the same time, the therapeutic effect of IFN-α+PD-1 blockade combination therapy on other types of tumors should also be explored
.

However, there is no doubt that the IFN-α+PD-1 blockade combined treatment plan launched by Academician Fan Jia/Zhu Di/Xu Yang's team is exciting and exciting! The results of their initial clinical trials and mouse animal models are also very significant and significant! It is hoped that the follow-up related clinical trials will be equally outstanding, making a landmark contribution to the immunological treatment of tumors, and bringing real hope to the majority of tumor patients! References: [1]Villanueva A.
Hepatocellular Carcinoma.
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2019;380(15):1450-1462.
doi:10.
1056/NEJMra1713263[2] Llovet JM, Kelley RK, Villanueva A, et al.
Hepatocellular carcinoma .
Nat Rev Dis Primers.
2021;7(1):6.
Published 2021 Jan 21.
doi:10.
1038/s41572-020-00240-3[3]Llovet JM, Castet F, Heikenwalder M, et al.
Immunotherapies for hepatocellular carcinoma .
Nat Rev Clin Oncol.
2022;19(3):151-172.
doi:10.
1038/s41571-021-00573-2[4]Qin S, Ren Z, Meng Z, et al.
Camrelizumab in patients with previously treated advanced hepatocellular carcinoma: a multicentre, open-label, parallel-group, randomised, phase 2 trial.
Lancet Oncol.
2020;21(4):571-580.
doi:10.
1016/S1470-2045(20)30011-5[5] Hu B, Yu M, Ma X, et al.