Main results
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement 795272.
Summary of the context and overall objectives of the project
Despite dedicating enormous research and socioeconomic efforts, cancer remains as the second cause of death globally. According to the World Health Organization, the number of new cases is expected to rise by about 70% over the next 2 decades, with 70% of deaths from cancer occurring in low- and middle-income countries. Furthermore, the economic impact of cancer is significant and is increasing, with an annual cost estimated at approximately 1.16 trillion $ in 2010. Among different tumor types, breast cancer has the highest incidence among women, causing 684996 deaths worldwide in 2020, and being the fourth cause of cancer related deaths. Thus, any improvement in cancer treatment will have a great impact in the health of millions of patients and will benefit the society as a whole.
In recent years, cancer immunotherapies have gained significant importance. These therapies are designed to activate the immune system against cancer cells. The field has been recently revitalized with the discovery of immune checkpoints such as programmed cell death protein-1 (PD-1) and its ligand (PD-L1). As the regulators of the immune system activation, these immune checkpoints are utilized by cancer cells to escape immune surveillance. Once PD-L1 binds to PD-1, the immune cell becomes deactivated inhibiting the natural immune response to cancer. Immune checkpoint inhibitor therapies, such as the use of anti- PD-1 or PD-L1 monoclonal antibodies, trigger an immune response against cancer cells by blocking immune checkpoints used by cancer cells to escape immune destruction. PD-L1 is often overexpressed in cancers and it has been successfully exploited for immune therapy in different tumor types, such as melanoma, non-small cell lung carcinoma and renal cell carcinoma. However, treatment outcomes have not been as effective in other cancers such as breast and pancreatic cancer.
The explanation for this lack of efficacy of cancer immunotherapies is multifactorial, including lack of immune cell infiltration in the tumoral mass, T-cell exhaustion and the development of acquired immune resistance. However, multiple studies point towards cancer aberrant metabolism as one of the key causes of cancer acquired immune resistance. Although most cancers exhibit an aberrant metabolism, one common feature shared by most of them is the increased levels of phosphocholine and total choline-containing compounds. These changes have been attributed, in large part, to the overexpression and increased activity of choline kinase (Chk)-α in malignant cells.
Thus, the main objective of ONCOTHERANOSTICS, my MSCA project, is to assess the mechanism of immunotherapies failure and to develop novel approaches that allow to overcome tumor resistance. This overall objective is divided in multiple specific objectives, two of them being already completed. The first specific objective is to investigate the molecular links between tumor metabolism, cancer acquired immune resistance and immunotherapies failure. For this objective, we have focused in Chk-α and PD-L1, as both are known to play highly significant roles in tumor aberrant metabolism and tumor acquired immune resistance, respectively. The second specific objective is to develop a novel theranostic agent that allow to selectively decrease the tumoral levels of PD-L1 in vivo. For that, we have developed a new nanoparticle (NP) based on the biodegradable polymer dextran. All these studies were perfomed in a model of Triple Negative Human Breast Cancer, the most aggressive form of breast cancer, and thus the most in need of new therapeutic alternatives.
Work performed and main results achieved so far
The activities described here were performed during the first period of ONCOTHERANOSTICS. This period include the first 24 months of the action and it has been developed in the Department of Radiology and Radiological Science, in The Johns Hopkins University School of Medicine, under the supervision of Professors Bhujwalla and Cerdan. During this period, the action was divided into two different specific objectives. The main activities developed and results obtained in these two objectives are the listed below:
Specific Objective 1: Study of the relationship between cancer aberrant metabolism and the expression of the immune checkpoint inhibitor PD-L1.
Work performed
These studies were carried in vitro using four different cancer cell lines, two triple-negative human breast cancer cell lines (MDA-MB-231 and SUM-149) and two human pancreatic ductal adenocarcinoma cell lines (Pa09C and Pa20C). We treat the cancer cell lines with small interfering RNA (siRNA) in order to decrease the expression of Chk-α, PD-L1, or a combination of both. The effects of the downregulation were studied at the:
1. Genomic level, using real time polymerase chain reaction (rt-PCR) techniques.
2. Proteomic levels, using western blot (WB) and flow cytometry (FC) techniques.
3. Metabolomic levels: in order to assess the metabolic changes induced by the siRNA treatment, cells were extracted using a dual phase methodology. These extracts were analyzed using high resolution magnetic resonance spectroscopy (HR-MRS) to measure the metabolic profile of the cells.
4. Inflammation: the production of the inflammatory molecule prostaglandin E2 (PGE2) in response to the treatment with siRNA was assessed using enzyme-linked immunosorbent assay (ELISA) techniques.
Finally, we looked for a clinical confirmation of the results. In order to accomplish that, the results obtained in vitro were compared with the results obtained through the analysis of public data from The Cancer Genome Atlas Program (TCGA).
Main Results
We identified an inverse dependence between Chk-α and PD-L1 at the genomic, proteomic, and metabolomic levels. We demonstrated that downregulation of Chk- α has a direct impact on the levels of PD-L1, but also that PD-L1 downregulation has a direct effect on cancer cell metabolomics. We also found that prostaglandin-endoperoxide synthase 2 (COX-2) and transforming growth factor beta (TGF-β) play an important role in this relationship. We independently confirmed this relationship in human cancers by analyzing data from The Cancer Genome Atlas Program. These results were published in a peer review article.
Specific Objective 2: Development and testing of a theranostic agent to deliver siRNA targeting PD-L1 in vivo.
Work performed
We developed a new nanoparticle (NP) as a theranostic agent to selectively deliver siRNA to cancer cells. The effectivity of this NP to deliver siRNA was tested in vitro and in vivo using the triple-negative human
breast cancer cell line MDA-MB-231. The following activities were performed:
1. NP synthesis and functionalization: we used the biodegradable molecule dextran as the scaffold for the NP. Dextran was functionalized with small molecules containing amine groups through acetal bonds used to bond the siRNA. The NP was decorated with a Cy5.5 NIR probe allowing visualization of NP delivery, accumulation, and biodistribution in vivo.
2. In vitro studies: cancer cells were treated with the PD-L1 siRNA NP complex. The cells were studied as detailed previously at the genomic and proteomic levels.
3. In vivo studies: MDA-MB-231 cancer cells were inoculated orthotopically or subcutaneously in the the mammary fat pad of mice. Animals were treated with two doses of PD-L1 siRNA NP complex. The biodistribution of the NP was studied in vivo and ex vivo using optical imaging approaches. The effects of the siRNA in the tumors were assessed as detailed in the previous section.
Main Results
We demonstrated that the PD-L1 siRNA dextran NP effectively downregulated PD-L1 in MDA-MB-231 cells. Using in vivo imaging, we demonstrated that the size of the NP of ~ 20 nm allowed delivery through leaky tumor vasculature but not through the vasculature of high PD-L1 expressing normal tissue such as the spleen and lungs. We identified a preferential accumulation of the NP in the tumor, finding a significant correlation between NP accumulation in the tumor, and the extent of PD-L1 downregulation. These results were published in a peer review article.
Progress beyond the state of the art, expected results and potential impacts
Specific Objective 1: Study of the relationship between cancer aberrant metabolism and the expression of the immune checkpoint inhibitor PD-L1.
Progress beyond the state of the art
Our data identified previously unknown roles of PD-L1 in cancer cell metabolic reprogramming, and revealed the immunosuppressive role of Chk-α downregulation through the increase of PD-L1. We demonstrated, for the first time, the direct involvement of Chk-α in immune suppression, as Chk-α downregulation significantly increased PD-L1 levels. Chk-α downregulation also shifted cancer cells towards a more immunosuppressive profile through metabolic reprogramming, increasing the production of metabolites such as lactate, glutamate, or glutamine, which have been linked to increased immune resistance of cancer cells. Downregulation of PD-L1 also resulted in a significant increase of immune-suppressive metabolites such as lactate, glutamate, and phosphocholine, although the number of metabolites that were altered was fewer compared to Chk-α downregulation. The significant increase of lipid production and changes in the lipid profile following PD-L1 downregulation may also contribute to cancer cells escaping immune surveillance. These findings are supported by several studies that have shown how lipids can reprogram tumor-infiltrating myeloid and T cells towards immunosuppressive and anti-inflammatory phenotypes.
PD-L1 downregulation may also create a more immunosuppressive profile through increased expression of TGF-β and COX-2, both of which are related to immune escape and anti-PD-L1 treatment failure. We detected increased TGF-β, COX-2, and lipid production with PD-L1 downregulation. Conversely, TGF-β and lipid production decreased following increased PD-L1 expression in response to Chk-α downregulation. These data support an active role of both TGF-β and COX-2 in the dependence between Chk-α and PD-L1. Furthermore, our results demonstrate, for the first time, that PD-L1 plays a significant role in COX-2 and TGF-β modulation in cancer cells.
Future Results
The metabolic reprogramming observed following downregulation of PD-L1 and its effects on the tumor immune microenvironment merit further investigation. This metabolic reprogramming that, based on our data, is mediated through Chk-α, may assist cancer cells in escaping immune surveillance in response to a decrease of PD-L1, or may be a component of the PD-L1 immune checkpoint program in activating the immune system. Further studies investigating metabolic reprogramming controlled by the different immune checkpoints will provide a more comprehensive understanding of the interaction between the immune checkpoints and metabolism. Also, the roles of the inflammatory transcription factor NF-kappa-B and hypoxia inducible factor 1 α regulation in mediating the interactions between Chk-α and PD-L1 will be investigated in future studies.
A major unmet need in treatment with immune checkpoint inhibitors is the lack of a noninvasive technique to identify patients who may benefit from such a therapy. Our results suggest that tumors with low PD-L1 expression may have high Chk-α expression and consequently high PC and total choline that can be detected noninvasively with MRS. Future studies relating total choline detected by 1H MRS in tumors to PD-L1 expression in biopsy samples may provide further evidence for the development of the total choline signal as a biomarker to predict for PD-L1 expression levels.
Potential impacts
The results presented have a great impact in the understanding of how different tumors acquired immune resistance and to immunotherapies failures. The increase of PD-L1 as a consequence of Chk-α downregulation, identified low Chk-α as contributing to immune suppression in cancer cells, different from its role as an oncogenic protein when overexpressed. Consequently, treatments that target Chk-α may result in cancer cells escaping immune surveillance. Also, the impact of PD-L1 in tumor metabolomics paves a new avenue to understand the failure of therapies based on antibodies targeting PD-L1. Our data also proved that PD-L1 regulation of metabolism may be mediated through Chk-α, COX-2, and TGF-β. These observations provide new insights that can be applied to the rational design of combinatorial therapies targeting immune checkpoints and cancer metabolism.
Specific Objective 2: Development and testing of a theranostic agent to deliver siRNA targeting PD-L1 in vivo.
Progress beyond the state of the art
Here we have demonstrated, for the first time, the feasibility of downregulating PD-L1 in tumors using siRNA delivered with a biodegradable dextran polymer that was decorated with an imaging reporter. Our data demonstrate the importance of tumor NP delivery and accumulation in achieving effective downregulation, highlighting the added value of imaging in siRNA NP delivery. Effective delivery of these siRNA-carrying NPs in the tumor but not in normal tissues may mitigate some of the side-effects of immune checkpoint inhibitors by sparing PD-L1 inhibition in these tissues.
Future Results
Multiple studies have demonstrated that PD-L1 has pro-oncogenic roles beyond its traditional functions in immunomodulation. Future studies will evaluate the impact of in vivo PD-L1 downregulation in tumor development, aggressiveness, and metastatic potential. Beyond this, our results and others studies have shown the impact of tumor metabolism on PD-L1 levels and the immune response. As a result, metabolic inhibitors of different pathways are being evaluated in clinical trials in combination with immune-checkpoint inhibitors, with promising outcomes. The new dextran NP creates the fascinating possibility of including siRNA that downregulate enzymes in metabolic pathways in combination with immune checkpoint siRNA.
Potential impacts
Antibody-based immunotherapies target normal tissues where the immune checkpoint is expressed along with the tumor. This leads to significant side-effects. As a result, novel approaches to targeting immune checkpoints using small molecules, peptides and macrocycles, are being actively explored. But inhibiting PD-L1 in organs that normally express PD-L1, as lungs, heart or colon, can lead to immune-related pneumonitis, myocarditis, and colitis. The use of siRNA NPs that primarily accumulate in tumors but not in normal tissues would reduce side-effects associated with immune checkpoint inhibition in normal tissues. In addition, the use of siRNA NPs provides the potential to combine multiple siRNAs directed toward different molecular pathways, including multiple immune checkpoints, within a single NP.