Precision medicine seeks to treat patients wi­th the right drug at the right time. In oncology, precision or “personalized” treatments are based on the specific biologic and biochemical characteristics of an individual’s tumor. Personalized treatment requires the development of in vivo cancer models that sufficiently represent those traits, and that enables distinguishing from among several genotypes or phenotypes. Using cells derived from biopsies, surgical resections, and autopsy tissues as their starting cultures, the Englander Institute for Precision Medicine at Weill Cornell Medical College has been developing organoids as next-generation cancer models.

To date, our efforts have produced 112 unique “next-generation organoids” representing eleven primary tumor types (colorectal, breast, lung, stomach, esophagus, ovary, endometrium, pancreas, kidney, bladder and prostate) that can be used for biological research and for high-throughput drug screening.

Three-dimensional (3D) cell culture has replaced 2D cultures for a variety of biological assays, particularly those that rely on replicating a cell’s in vivo microenvironment. Spheroids and organoids are the principal 3D culture methods. Although the terms are sometimes used interchangeably, spheroids typically consist of suspended, free-floating cell aggregates while organoids are more complex higher-order structures generated through self-organization within an extracellular matrix, a vital component of growing such models. Organoids tend to be “grown” from induced pluripotent stem cells, isolated organ-progenitor cells from adult tissues[1], or from cancer stem cells, which have the capacity for infinite expansion. The Englander Institute for Precision Medicine’s (EIPM) location at a major metropolitan research hospital enables the opportunity to design cancer organoids from nearly every tumor phenotype.

When derived from stem- or stem-like progenitors that are appropriately differentiated, organoids can form wild-type structures that faithfully recreate the function and characteristics of normal cells in healthy tissues. These 3D cultures serve as models for drug screening and toxicology studies and can be manipulated to mimic disease states as well.

Patient-derived tumor organoids have filled the gap between 2D cell lines and xenografts of human tissues in animal models, a technique that is decades-old, with the advantages of taking less time, not requiring an animal facility, and the ability for mass-production. This advancement further catalyzes the shift toward precision medicine and underscores the importance of creating consistent conditions for organoid formation.

Organoids and Precision Oncology

Personalized or precision oncology has grown from the realization that tumor appearances can be deceiving—treating all histopathologically “identical” tumors the same way while ignoring cancer subtypes or genetics has failed to improve outcomes for cancer patients.[1] Popular methods of drug discovery haven’t yet achieved the necessary efficiency to benefit the general patient. Even gene sequencing and 3D cultures are too time and resource-intensive to be generally useful.[2],[3]

By contrast, organoids derived from recently harvested tumors can be expanded into organ-like structures to generate patient-specific cancer models in several weeks. As cells within organoids expand and multiply, they self-assemble into structures resembling in vivo tissues, and in some instances take on in vivo phenotypes. By thus recapitulating the tumor epithelial microenvironment, including the potential for cell-cell interactions, organoids come closer than any other cell culture technique in providing an appropriate biological niche for exploring a patient’s treatment options from among dozens of approved and experimental cancer drugs. Tumor-derived organoids may subsequently be used to test the susceptibility of that particular patient’s cancer to a range of monotherapies or combination treatments, to provide guidance on prognosis, and in some instances to monitor disease progression.

Cancer cell lines and patient-derived xenograft models are widely used in drug screening and to assess or predict a tumor’s response to treatments. Cancer cells multiply rapidly relative to healthy somatic cells, and they are amenable to genetic manipulation that creates specific sub-phenotypes.[1] A drawback of cultured cancer cell lines is the genetic and histologic drift these cells undergo in culture, particularly after many passages.[2] It comes then, at no surprise, that many drugs that work on cultured cancer cells fail in clinical testing. With respect to personalized or precision therapies, tumor-derived organoids retain the genomic and transcriptomic characteristics of the original tumor.[3] Therefore, responses to treatment relevant to safety and efficacy may be compared, side-by-side, with organoids derived from healthy cells harvested from the same patient.

Despite their current popularity as alternatives to 2D cultures and xenograft models, tumor-derived organoids are not perfect disease models. They lack blood vessels, whose formation in the vicinity of tumors is a known factor in tumor metastasis and nutrition.[4] Drugs tested on organoids must, therefore, diffuse into the cell masses rather than entering through circulation. Moreover, organoids derived from tumor cells alone lack the stromal and immune system components that often affect the course of cancer. Organoid technology is now evolving to incorporate components of the microenvironment that would expand the type of therapies that can be tested.[5]

Growing Consistent Organoids: Workflow Brought to Life

Tumor organoid workflows begin with selected patients undergoing biopsies or tumor resection and subsequently referred for clinical genomic follow-up. Working with a major metropolitan teaching hospital affords us the opportunity to gain exposure to a wide variety of diseased tissues.

Samples positive for tumors of interest enter the culture phase, where they undergo at least five passages followed by biobanking and verification through automated RNA sequencing, next-generation sequencing, cytology, and immunohistochemistry. Cells in excess of our immediate needs are frozen and stored for later use; for example, if organoid culturing fails at a later passage. Passage time varies for different tumor types, but one to three weeks is typical.

Since the goal is to create organoids that recapitulate the cellular heterogeneity of the original tumor, we make no attempt to enrich or deplete samples for any specific type of cell.

From there, the organoids may be used like any other cell culture, including for drug screening, optimization of culture conditions (e.g. extracellular matrix or microenvironment), or for personalized diagnostics/precision medicine.

Sample preparation is straightforward. Tissues are first reduced in size manually, then cells are freed from their matrix using collagenase to create a suspension of single cells or clusters of cells. Cells are washed with a cell culture medium-high in vitamins and amino acids that serve as the organoid growth medium. At this stage, cells are suspended in an appropriate extracellular matrix to provide an in vivolike niche for tumor cell expansion and organoid self-assembly.

Many components of our “typical” organoid workflows have been optimized specifically for tumor-derived organoids and some are interchangeable and generic (i.e. tissue mincing or cell detachment). While little can be done to minimize the variability of the tissue source, including the tumor’s inherent inhomogeneity, the goal is nevertheless to produce organoids that behave consistently under similar experimental conditions, between batches, experimenters, and even laboratories. The choice of the extracellular matrix is critical towards achieving that level of consistency.

Selecting Methods for Success

In surveying methods suitable to large-scale production of tumor-based organoids, it soon became apparent that Corning® Matrigel® matrix, with more than 10,000 scientific citations, is by far the most-used in vivo extracellular matrix (ECM).[6] It has been known since the early 2000s that Matrigel matrix, a gelatinous protein mixture derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, contains several components (laminins, collagen IV, enactin, heparin sulfate, and growth factors) that are highly representative of tumor microenvironments.[7]

Corning, the manufacturer of the Matrigel matrix, has invested considerable resources into standardizing the physical and mechanical properties of the matrix. This is particularly useful for 3D cell culture and now there is a Matrigel matrix for organoid culture, an optimized version of the ECM.

Conclusion

With the occurrence of irreproducible results in the life sciences presenting ongoing challenges, consistency and reproducibility have assumed critical importance.[8] So important, in fact, that institutions like EIPM have partnered with the National Cancer Institute to generate and collect culture models and their respective genetic data as a community-owned resource.[9]

As interest in organoids as disease models heighten, researchers will increasingly rely on robust, validated methods. In this environment, investigators will insist on the same level of rigor and consistency from their suppliers, particularly for products that come into contact with cells. For 3D cell cultures, in general, “the niche is the product,” which includes culture media, growth factors, feed supplements and—above all—the extracellular matrix. Only through attention to these details will tumor-derived organoids thrive as a field and reach a level of industrialization commensurate with their growing importance in cancer research.

 

Laura Martin, PhD, is the ex vivo models director at the Englander Institute for Precision Medicine (EIPM) within Weill Co­­rnell Medicine. Elizabeth Abraham, PhD, is a senior product manager at Corning Life Sciences.

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