*3.4.2. Orthotopic xenografts*

A number of observations suggest that the behavior of tumor cells can be significantly dif‐ ferent when implanted as a subcutaneous xenograft, compared to their behavior when grown into the tissue of origin. For these reasons, orthotopic models are thought to be better predictors of drug efficacy and are more clinically relevant. To this purpose, intracolonic xenografts have been developed. Technically, a small incision is made in the abdomen of the immunodeficient mouse, directly over the colon, and CRC cells are implanted under the se‐ rosa of the colon. Local tumor growth on the colon is then monitored. Although more realis‐ tic, the use of orthotopic xenograft models does not guarantee success. The efficacy of Semaxanib, an antiangiogenic molecule, has been tested in preclinical stages using an intra‐ colonic Xenograft [73] but compound development was stopped after negative results from Phase III. Again, representation of patient heterogeneity should be taken into account at the preclinical level.

Finally, if the use of selected tumor lines and the value of the mouse as a host could be ques‐ tionable in xenograft models, the response end points, survival end points, and tumor cell killing end points that are usually used during *in vivo* efficacy studies remain in line with clinical investigations.

#### *3.4.3. Patient-derived xenograft models (PDXs)*

In order to circumvent the difficulties of establishing new cell lines, as well as to establish an *in vivo* model preserving the histopathological characteristics of the original tumor, investi‐ gators have developed a new xenograft system based on the direct grafting of human tumor fragment into immunodeficient mice (Figure 4). Several CRC patient-derived xenograft col‐ lections (PDX) have been reported, with an average tumor take rate of over 60% [56, 74-75]. They can be cryopreserved and re-established in mice as needed, or maintained as xeno‐ grafts from mice to mice. Intensive characterization has demonstrated that the architecture of PDX tumors, their gene expression profile and their chromosomal instability remains very similar to the parental tumor, even after successive passages [75-76]. Importantly, high correlation between drug activity in PDX and clinical outcome has been reported, making this model a valuable pharmacological tool for drug development [74-75]. Moreover, be‐ cause they are derived from tumor fragment, PDX tumors retains the genetic heterogeneity existing in the original human tumor and are therefore useful for studies exploring acquired drug resistance mechanisms [75, 77]. The use of PDX as a model for tumor-stroma interac‐ tion is however less obvious since by the fourth passages human tumor stroma is replaced by the murine host [75]. All together, the above considerations highlight the potential of the PDX model to accelerate drug development and predictive biomarker discovery in CRC.

**Figure 4.** The PDX model. Sequential steps leading to the establishment of a CRC primary Patient-Derived Tumor Xen‐ ograft collection. Briefly, a CRC tumor fragment coming from surgical waste is directly xenografted in an immunodefi‐ cient mouse (Passage 0). After successful engraftment, new fragments are taken from the mouse hosted human tumor and xenografted again in multiple immunodeficient mice (Passage 1). A collection of fragments from the re‐ sulting tumors can then be cryopreserved in a tissue bank for subsequent experiments or directly re-engrafted in mice for expansion (P2, P3, etc…). At any step, tumor fragments can be analyzed and compared to the parental tumor in

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As previously mentionned, current 2D monolayer culture systems are not enough predictive of *in vivo* tumor behavior. Indeed, 3D environement is required to provide essential signal‐ ing necessary for establishing and maintaining tumor specific morphogenic programs. Thus,

terms of gene expression, genetic mutations, genomic stability or histopathological features.

**3.5***. Ex-Vivo* **Organotypic Culture models (EVOCs)**

Colon Cancer: Current Treatments and Preclinical Models for the Discovery and Development of New Therapies http://dx.doi.org/10.5772/53391 449

**Figure 4.** The PDX model. Sequential steps leading to the establishment of a CRC primary Patient-Derived Tumor Xen‐ ograft collection. Briefly, a CRC tumor fragment coming from surgical waste is directly xenografted in an immunodefi‐ cient mouse (Passage 0). After successful engraftment, new fragments are taken from the mouse hosted human tumor and xenografted again in multiple immunodeficient mice (Passage 1). A collection of fragments from the re‐ sulting tumors can then be cryopreserved in a tissue bank for subsequent experiments or directly re-engrafted in mice for expansion (P2, P3, etc…). At any step, tumor fragments can be analyzed and compared to the parental tumor in terms of gene expression, genetic mutations, genomic stability or histopathological features.

#### **3.5***. Ex-Vivo* **Organotypic Culture models (EVOCs)**

able to recapitulate the biological heterogeneity of patient's populations appear necessary

A number of observations suggest that the behavior of tumor cells can be significantly dif‐ ferent when implanted as a subcutaneous xenograft, compared to their behavior when grown into the tissue of origin. For these reasons, orthotopic models are thought to be better predictors of drug efficacy and are more clinically relevant. To this purpose, intracolonic xenografts have been developed. Technically, a small incision is made in the abdomen of the immunodeficient mouse, directly over the colon, and CRC cells are implanted under the se‐ rosa of the colon. Local tumor growth on the colon is then monitored. Although more realis‐ tic, the use of orthotopic xenograft models does not guarantee success. The efficacy of Semaxanib, an antiangiogenic molecule, has been tested in preclinical stages using an intra‐ colonic Xenograft [73] but compound development was stopped after negative results from Phase III. Again, representation of patient heterogeneity should be taken into account at the

Finally, if the use of selected tumor lines and the value of the mouse as a host could be ques‐ tionable in xenograft models, the response end points, survival end points, and tumor cell killing end points that are usually used during *in vivo* efficacy studies remain in line with

In order to circumvent the difficulties of establishing new cell lines, as well as to establish an *in vivo* model preserving the histopathological characteristics of the original tumor, investi‐ gators have developed a new xenograft system based on the direct grafting of human tumor fragment into immunodeficient mice (Figure 4). Several CRC patient-derived xenograft col‐ lections (PDX) have been reported, with an average tumor take rate of over 60% [56, 74-75]. They can be cryopreserved and re-established in mice as needed, or maintained as xeno‐ grafts from mice to mice. Intensive characterization has demonstrated that the architecture of PDX tumors, their gene expression profile and their chromosomal instability remains very similar to the parental tumor, even after successive passages [75-76]. Importantly, high correlation between drug activity in PDX and clinical outcome has been reported, making this model a valuable pharmacological tool for drug development [74-75]. Moreover, be‐ cause they are derived from tumor fragment, PDX tumors retains the genetic heterogeneity existing in the original human tumor and are therefore useful for studies exploring acquired drug resistance mechanisms [75, 77]. The use of PDX as a model for tumor-stroma interac‐ tion is however less obvious since by the fourth passages human tumor stroma is replaced by the murine host [75]. All together, the above considerations highlight the potential of the PDX model to accelerate drug development and predictive biomarker discovery in CRC.

for an accurate evaluation of molecular targeted agents.

*3.4.2. Orthotopic xenografts*

448 Drug Discovery

preclinical level.

clinical investigations.

*3.4.3. Patient-derived xenograft models (PDXs)*

As previously mentionned, current 2D monolayer culture systems are not enough predictive of *in vivo* tumor behavior. Indeed, 3D environement is required to provide essential signal‐ ing necessary for establishing and maintaining tumor specific morphogenic programs. Thus, an *ex vivo* methodology which can recapitulate physiological processes and generate multi‐ ple experimental replicates from a single tumor, saving at the same time animals involved in *in vivo* experiments will be of great benefit. *Ex vivo* organotypic cultures (EVOCs), by pre‐ serving the original cancer microenvironement (e.g. epithelial-stromal interaction) fulfill this requirement. Recently, a number of culture methods have been perfected leading to the de‐ velopment of breast, lung, liver and colon EVOC tumor models [78-81]. EVOCs allow the evaluation of tumor morphology, proliferation, viability and resistance to therapy *in vitro*. Moreover, differential gene-expression profiling across tumor and stroma compartments can be performed, without any contamination coming from a murine host as seen in xeno‐ graft models [78]. Recent observations have shown that CRC EVOCs mimic closely the *in vivo* situation, at the immunohistochemical level [81], but also in term of oncogenic pathway fonctionallity and pharmacodynamic properties [78]. Importantly, dose-response experi‐ ments with the PIK3 inhibitor LY294002 demonstrate that CRC EVOCs may be used to pre‐ dit tumor sensitivity to drugs in a patient-specific manner [78]. EVOCs represent therefore a highly promising *in vitro* tumor model, when combined with automated medium-through‐ put analyses, has the potential to significantly enhance preclinical drug evaluation studies.

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