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Lyons et al. Cancer Drug Resist 2021;4:745-54 https://dx.doi.org/10.20517/cdr.2021.37 Page 747

ADVANCES IN PRECLINICAL CANCER MODELS
The past decade has been transformative for tissue culture technology of patient-derived tumors. Until
recently, only a limited number of immortalized 2D cancer cell lines was available to test the preclinical
performance of an ADC using xenograft mouse models. Such models remain popular today as they are
relatively quick and easy to develop. The cell lines are widely distributed among the research community,
[7]
and some have been the focus of extensive genomic and gene expression characterization . However, such
cancer models also have significant deficiencies, can be prone to genetic drift over time, and their ability to
accurately model human disease and ultimately predict the clinical performance of candidate therapeutics is
questionable. The failure rate of establishing an immortalized tumor cell line in culture by traditional means
is extremely high. Human tumors did not evolve to grow on tissue culture plastic as a 2D monolayer; thus, it
is questionable how representative the low frequency of successfully established cultures are of the original
tumor. Further, although subcutaneous engraftment of such cells in vivo is very routine (quick to establish
and tumor development is externally visible), such models fail to recapitulate the tumor microenvironment
that matches their native tissue of origin.

The advent of patient-derived organoid (PDO) cell cultures has set new biologically relevant standards that
overcome many limitations of conventional 2D xenograft cell lines . Tumor samples received fresh from
[8]
the operating theatre are processed and plated out in vitro in a mixture of growth factors and basement
membrane extract such as Matrigel (a viscous matrix resembling a decellularized tissue microenvironment)
to provide more natural growth conditions for the cells. As a result, PDOs grown in three dimensions in
vitro retain cell polarity and some semblance of structure that can provide biologically relevant cues
[Figure 1]. The efficiency and general applicability to various tumor types also far surpass that of traditional
2D methods. The success rate of establishing a PDO culture can be as high as 75% for tumors from a wide
range of epithelial organs. Once established, they can be propagated in vitro with relative ease in defined
culture media for extended periods of time. Analyses suggest that organoid cultures are relatively stable
genetically for many passages [9,10] , which opens up a host of research possibilities regarding their genetic
modification and experimental possibilities, as discussed in Sections ii and iii. Another ground-breaking
feature of this approach is that it is possible to establish organoid cultures from matching normal, tumor,
and metastatic tissue from the same individual. This is of crucial experimental importance given the outbred
nature and diverse genetic background of the human population. Moreover, organoids derived from
sequential specimens from the same patient have been shown to recapitulate identical sensitivities and
[11]
resistance to treatment, as observed in the clinic .
The Human Cancer Model Initiative (an NIH/NCI-funded project ) was established as a novel resource to
[12]
give researchers access to these next-generation models via the American Type Culture Collection. Many of
the organoid lines available in this ground-breaking biorepository are also documented with patient and
sequencing information. Having such a well-characterized portfolio of cells enables many opportunities for
high-quality experimentation to evaluate ADC performance that was impossible or very hard to accomplish
in the past. For example, genetically and disease-matched PDOs can be used to assess the cytotoxicity of
targeted ADCs in vitro or in vivo and be compared to matching normal tissue-derived PDOs. As discussed
in Section iii, the conjugation of different moieties for imaging can be used to visualize where ADCs
accumulate naturally in the body and whether, once bound to the antigen, they stay on the surface of a cell
or are internalized.

PDO co-cultures can also be established in vitro to examine critical tumor cell interactions with defined
aspects of the tumor microenvironment, such as cancer-associated fibroblasts [13,14] , and possibly also with
immune cells in the near future. Such co-cultures will enable testing of an ADC on tumor and normal cells