New Tools Support Translational Strides in NSCLC

In non–small cell lung cancer (NSCLC), single cell analytics, genome editing, and next-generation animal models represent just some of the modern modalities advancing translational research, according to Charles M. Rudin, MD, PhD.¹

“[These are] the tools that are emerging as technologies that are enabling us to really dig into cancer biology in a new way,” Rudin said during the keynote address at the 21st Annual International Lung Cancer Congress^®, hosted by Physicians’ Education Resource^®, LLC (PER^®). “Single cell analytics is a huge field now that gives us insight into tumor heterogeneity, the evolution of tumors, and the immunologic dynamics.”

Beyond single cell analytics, which allow translational researchers to examine the immune composition of the tumor and how it changes over time, gene editing has also revolutionized efforts to discover new targets and create multigene synthetic lethality, according to Rudin. Of the gene editing techniques currently in use, clustered regularly interspaced short palindromic repeats (CRISPR) has been particularly impactful, he said. CRISPR are genomic sequences that bacteria use to recognize and destroy foreign DNA through an enzyme, such as the CRISPR-associated (Cas) protein, Cas9, which is guided by an RNA molecule. The recent uptake of CRISPR-Cas9 in oncology can be credited to its facilitation of more rapid, cost-effective, efficient, and specific gene editing than other existing methods.² The CRISPR-Cas9 technology has been “transformative” in the oncology space, Rudin said.

Rudin specified next-generation animal models as another tool critical to translational research. “These models have emerged in the past several years and are allowing us to do much more refined target validation and pharmacogenomic testing,” added Rudin, who is chief of Thoracic Oncology Service and codirector of the Druckenmiller Center for Lung Cancer Research at Memorial Sloan Kettering Cancer Center in New York, New York.

Single Cell Analytics

In the past, single cell profiling in NSCLC entailed RNA sequencing that assessed the whole tumor. Single cell analytics, in contrast, encompasses dissecting the composition of the tumor, Rudin explained. “When we think about single cell analytics, we’re [asking], ‘What are the individual cell types that make up that tumor, what is the stromal composition?’” 

Spatial trancriptomics and other spatial imaging approaches enable researchers to obtain answers to these clinical inquiries. “These [methods] allow us to look at a single cell basis at not just who’s in the tumor, but who’s talking to who within the tumor; what the tumor looks like both in terms of gene expression and in terms of interactions,” Rudin said.

Single-cell examinations can be done at transcriptional and proteomic levels to provide new insight into the heterogeneity of tumors and the biology of tumor development. Consequently, these novel spatial transcriptomics and spatial imaging modalities are quickly changing the way that oncology experts think about cancer biology.¹

CRISPR Technology and Next-Generation Animal Models

CRISPR technology, especially CRISPR-Cas9, has been of increasing interest in recent years and continues to inform translational research using CRISPR-edited mouse models in NSCLC. “This has just exploded as a field [given] the ability [it affords] to model genomic alterations and driver mutations, knock out genes, and do gene editing in vivo to study cancer biology,” Rudin said.

Rather than develop transgenic mouse models, which involves mating and crossing to get 2 alleles in a given mouse, investigators are using CRISPR technology to perform gene editing in the embryonic stem cells of mice. This methodology allows for quicker modeling of translocations and driver alterations.¹ 

“If you want to model 3 different genes [in a mouse], it [can be] a really complicated cross. With CRISPR, we can do them all with gene editing in the stem cell and then let the mouse grow in a single generation and basically generate any mouse that we may want to create relatively quickly,” Rudin said. “The ability to generate triple, quadruple, and higher-order alterations within the mouse [supports] modeling more complex alterations and multiple genes within genetically-engineered mouse models.”

To facilitate gene editing via CRISPR-Cas9, researchers create a piece of RNA with a guide sequence that binds to a prespecified target sequence of DNA in a genome and the Cas9 enzyme. The modified RNA recognizes the DNA sequence and the Cas9 enzyme cuts the DNA at the specified location. When the DNA has been severed, investigators use the cell’s DNA repair machinery to add or delete pieces of genetic material or make changes to the DNA. These alterations typically include replacing an existing segment with a customized DNA sequence.³

Developing the pSpCTRE System

Patient-derived xenografts (PDXs) have been of great interest to Rudin and colleagues given their high-fidelity reflection of several key aspects of human cancers.^1,4 In a patient-derived model, a patient’s tumor biopsy is implanted into a mouse. A newer patient-derived approach, circulating tumor-derived xenograft, entails collecting circulating tumor cells present in a patient’s blood and directing these cells to the mouse via infusion.¹ 

Of note, PDXs are limited because they are not candidates for gene editing. “They are the way they are; there’s no way to edit them because they’re maintained in vivo,” Rudin said. Specifically, the continuous in vivo passaging of PDXs obstructs the use of antibiotic election methods used by current CRISPR-Cas9 systems. For this reason, CRISPR-Cas9 technology has been restricted to xenografts of human and mouse cell lines cultured extensively in vitro of genetically engineered mouse models.⁴ 

To address this limitation, Rudin and colleagues developed a method for CRISPR-Cas9 editing of PDXs using a tightly regulated, inducible Cas9 vector that does not require in vitro culture for transduced cell selection.⁴ Together, Rudin and fellow investigators determined that using a vector with a selectable marker and inducible Cas9, followed by doxycycline, single-cell dissociation, quick-spin transduction, and implantation into the next mouse results in tumor growth that can be selected in the next round for the expressing cells of interest.¹ “[As a result] we’ve got a tumor that’s 100% positive for this vector, where we can induce Cas9 and then introduce guides into that tumor in the same way when we passage with 1 animal,” Rudin said.

The method developed by Rudin and his research team is called Streptococcus pyogenes Cas9 Tet Response Element (pSpCTRE). pSpCTRE is an all-in-one-dox-inducible Cas9 lentiviral vector tailored for use in PDXs, which have grown increasingly integral to cancer research. The pSpCTRE system is expected to advance understanding of NSCLC biology and support the development of new therapeutic strategies in this disease setting.⁴

References

1. Rudin CM. Changing translational research landscape in NSCLC: future prospects. Presented at: 21st Annual International Lung Cancer Congress^® hosted by Phyisicans’ Education Resource^®, LLC (PER^®); July 23-25, 2020; Virtual. Accessed July 23, 2020.
2. CRISPR: genome editing comes of age. National Cancer Institute. Published September 23, 2015. Accessed July 23, 2020. https://www.cancer.gov/about-cancer/causes-prevention/research/crispr#1 
3. What are genome editing and CRISPR-Ca9? Genetics Home Reference. Published July 7, 2020. Accessed July 23, 2020. https://ghr.nlm.nih.gov/primer/genomicresearch/genomeediting
4. Hulton CH, Costa EA, Shah NS, et al. Direct genome editing of patient-derived xenografts using CRISPR-Cas9 enable rapid in vivo functional genomics. Nat Cancer. 2020;1:359-369. doi:10.1038/s43018-020-0040-8