Available mouse models differ in their ability to support the engraftment of functional human immune systems. Furthermore, the nomenclature surrounding the various strains and models is complex, as investigators are the first to acknowledge. A real game changer in the use of mouse models, says Michael Seiler, PhD, vice president, commercial products, Taconic Bioscience, is the ability to combine key elements of a human immune system with PDX models to enable the assessment of novel immunomodulatory and other agents that affect tumor response in the context of a functioning immune system.
But immuno-oncology therapies have moved into several new classes of treatment, including monoclonal antibodies mAbs , tumor vaccines, bispecific antibodies bsAbs , and chimeric antigen receptor CAR T cells. It does not recognize the mouse PD-1 receptor, which limits the utility of a traditional mouse at a crucial stage in drug development.
Regarding the increasing complexity of mice required for testing immuno-oncology agents, Edgar Wood, PhD, senior research director, oncology, Charles River Discovery Services, explains that for testing standard cancer drugs, like cytotoxic or targeted therapies, the basic platform has been human tumor material grown as a xenograft in immunodeficient mice.
He cautions, however, that research into human-specific immunomodulators requires two xenografts in mice—the tumor material plus the human immune cells. According to Wood, if you are hoping to modulate other cell types, such as myeloid cells or natural killer NK cells, PBMC engraftment may not represent the desired biology. Wood adds that for therapies that recognize the mouse counterpart of a target, traditional mouse syngeneic tumor models offer a fully intact mouse immune system and a variety of tumor types and responsiveness to immunomodulators.
James G. It is used to engraft human umbilical cord stem cells for the development of a partial human immune system in the mouse.
Building Better Mice for Cancer Drug Discovery and Development
The mice are healthy and robust, and live for about a year. The Jackson Laboratory plans to make additional mouse models that will, upon engraftment with umbilical cord stem cells, lead to additional human immune cell populations and allow researchers to ask more specific questions about the immune response to tumors. For these reasons, US and European Union have implemented preventive screening programs that have contributed to slightly reduce morbidity and mortality [ 10 ].
Unfortunately, as in many other forms of cancer, colon cancer does not display too many symptoms, develops slowly over a period of several years, and only manifests itself when the disease begins to extend. Adjuvant chemotherapy in combination with surgery or radiation is then the usual treatment.
However, 5 of the 9 anti-CRC drugs approved by the FDA today are basic cytotoxic chemotherapeutics that attack cancer cells at a very fundamental level i. These figures underline the urgent need to expand the standard therapy options by turning to more focused therapeutic strategies. In recent years, combination of basic chemotherapies with targeted therapies, in the form of humanized monoclonal antibodies directed against the vascular endothelial growth factor VEGF Bevacizumab to prevent the growth of blood vessels to the tumor, or directed against the EGF receptor Cetuximab, Panitumumab to block mitogenic factors that promote cancer growth, have been introduced as possible therapeutic protocol and used routinely to treat standard CRCs, as well as metastatic CRCs Table 1.
During the preparation of this manuscript August , another recombinant protein active against angiogenesis, Aflibercept, has been approved by the FDA for the treatment of metastatic CRC in second-line therapy Table 1. This new VEGF inhibitor has demonstrated a significant advance over currently available therapy in a Phase III study improvement in response rate and in overall survival; [ 11 ]. Accordingly, the expansion and the development of new path of therapy, like drugs specifically targeting the self-renewal of intestinal cancer stem cells - a tumor cell population from which CRC is supposed to relapse [ 12 ] — remains relevant.
Anti-cancer colorectal drugs approved by the Food and Drug Administration. Drugs are presented sorted by type, i.
Source: National Cancer Institute database, A classical approach of drug design in oncology is to identify modulators of specific signal transduction pathways that are important for tumor growth, survival, invasion, and metastasis. Many experiments have demonstrated that disruption of the WNT signaling pathway lead to consistent growth inhibition and apoptosis of CRC cell lines and effective inhibition of tumor growth in CRC animal models.
These results can be achieved by modulating the pathway at different levels, from the membrane receptor to the final nuclear transcription factors Figure 3. It is now well documented that a number of critical pathways regulating stem cell maintenance and normal developmental processes e. However, currently few of these compounds have progressed beyond the preclinical stages. KLF4 Kruppel-like factor 4 is a tumor suppressor factor which is typically deficient in a variety of cancers, including colorectal cancer. Nonetheless, considering the huge effort done at the research level to identify potential antagonists and the few candidate already engaged into preclinical studies, no doubt that innovative therapies will emerge from this promising pathway in a near future.
These observations pinpoint one of the most challenging aspects of anticancer therapy that is intrinsic or acquired drug resistance. Indeed, several studies have shown that these mutations are associated with the lack of response to Cetuximab and Panitumumab anti-EGFR therapies observed in a subset of chemorefractory metastatic CRCs, suggesting that the corresponding deregulated signaling pathways are responsible for the occurrence of resistance of the tumor to the clinical treatment [ 27 - 28 ].
As a result, downstream key components mostly protein kinases of these constitutively activated growth-related signaling cascades have become targets for drug development. This new class of drugs appears therefore as a promising third-line therapeutic strategy for colon cancer patients, especially after recurrence of tumor resistance. Depending on the tumor status, from pro-apoptotic tumor suppressor, PI3K or AKT inhibitors could become metastatic inducers [ 32 ].
All together, the complexity of these results supports the arrival of a personalized medicine, where a careful profiling of tumors will be useful to stratify patient population in order to test drugs sensitivity and combination with the ultimate goal to make treatments safer and more effective. As previously mentioned, until recently the humanized monoclonal antibody Bevacizumab against VEGF was the only anti-angiogenesis agent approved by FDA. It is now completed by Aflibercept, a recombinant protein consisting of the key domains of VEGF receptors 1 and 2.
Due to improvement in the understanding of the critical role of angiogenesis in the maintenance of CRC tumors and the spread of their metastasis, anti-angiogenesis has become an area of active investigation [ 35 ]. However, the recent failure in Phase III first-line studies of two promising compounds Sunitunib in and Cediranib in has cast serious doubt on that strategy.
Therefore, the approval of Aflibercept provides timely support to the further development of anti-angiogenics as treatment for metastatic CRC. Today, 4 additional therapeutic agents that target VEGF, Ramucirumab [ 36 ], Icrucumab [ 37 ], Regorafenib [ 38 ] and Vatalanib [ 39 - 40 ] are under clinical evaluation Table 2. This battery of anti-angiogenics is supplemented by AMG, a recombinant peptide-antibody fusion protein peptibody which targets another signaling pathway involved in tumoral angiogenesis, the angiopoietin axis [ 41 ].
TAS mechanism of action is based on the inhibition of the thymidine phosphorylase TYMP also known as the platelet-derived endothelial cell growth factor, a potent angiogenic factor [ 42 ]. In this context, it is important to point out that differences in the efficiency to block angiogenesis and tumor progression have been observed between preclinical models and clinical trials, when comparing antibodies with small molecules [ 35 ]. These discrepancies in clinical outcome underline the necessity to validate compounds on relevant models, preferentially based on human tissues, very early during drug development process.
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Modifications in the epigenetic landscape are commonly associated with cancer, but on the contrary to genetic mutations, these changes are potentially reversible and therefore druggable. Most of the epigenetic drugs discovered to date modulate DNA methylation or histone acetylation. Four epigenetic drugs have already been approved by FDA for use in clinic against various cancers.
Anti-cancer drugs in colorectal clinical trials. This table gives an overview of the main colorectal cancer therapies being currently evaluated in clinical trials. For each compound, the pathway target and clinical status is provided. Oncolytic viral therapy represents an appealing alternative therapeutic strategy for the treatment of CRC, both as single agent or in combination with existing clinical regimens. Oncolytic viruses, like the vaccinia virus a virus previously used for worldwide vaccination against smallpox , have the property to selectively infect and destroy tumor cells with limited or no toxicity to normal tissues.
These viruses efficiently replicate in tumor tissue, cause tumor lyses and stimulate antitumor immune response. During the last decade, numerous mutants have been engineered to improve their tumor specificity and antitumor efficacy, and to allow tracking of viral delivering by non-invasive imaging [ 44 ].
No less than five oncolytic virotherapies are currently evaluated in clinical trials for metastatic CRC indication, including ColoAd1, derived from an adenovirus [ 45 ], NV, derived from an Herpes simplex virus [ 46 ], Reolysin, a reovirus [ 47 ], and JX [ 48 ] and GL-ONC1 [ 49 ] both derived from vaccinia viruses, reflecting the many hopes carried by this emerging treatment modality.
However, it is noteworthy to mention that there are still some difficulties to viral infection. Solid tumors have a complex microenvironment that includes disorganized surrounding stroma, poor vascular network as well as high interstitial fluid pressure. All these parameters will limit viral delivery since viral penetration directly depends on cellular packing density and adhesion between cancer cells [ 50 ]. Moreover, hypoxia reduces viral replication, and therefore oncolytic efficiency, without affecting tumoral cells viability [ 51 ].
These observations highlight how choosing the right experimental validation model, e. This inventory of new drugs for the treatment of colorectal cancer highlights the diversity of approaches being considered to combat the disease. Whether based on small molecules, humanized antibodies or modified viruses, their success in further clinical assessment is largely related to the quality of their preclinical evaluation.
This is why both the choice of appropriate existing model systems and the development of more clinically relevant and predictive pre-clinical models appear critical in overcoming the high attrition rates of compounds entering clinical trials. Current research is also focusing on the development of biomarkers that will be useful for the early detection of CRC, as well as for fine-tuning drug regimen and following efficacy during trials and treatments.
To date, only a few markers have been recommended for practical use in clinic [ 52 ] but large-scale genomics technology combined with advanced statistical analyses should generate soon new biomarker panels for CRC diagnosis [ 53 ].
Then, it will be interesting to see how these biomarkers could be implemented in preclinical stages to improve drug selection. It is worth mentioning that most of our understanding of the molecular mechanisms involved in CRC come from studies done on mouse or human cell lines that represent only a highly selected fraction of the original tumor and that may have acquired in vitro additional genetic abnormalities.
Clearly, the scientific community has taken into account these limitations, as shown by the growing interest for more complex models e. However, although imperfect, colon cancer cell lines still represent a unique resource that can be extremely valuable in term of genetic manipulation and high-throughput screening, with cell viability, cell proliferation or promoter specific reporter activity being the usual endpoints followed.
Several initiatives have been launched to maximize their utility in large scale drug discovery programs. In an attempt to identify new active molecules, over , chemical compounds were pharmacologically tested in this cell line set. But disappointingly, most of the selected positive candidates were typical cytotoxics, affecting cancer cells via general fundamental cellular processes, like cell cycle regulation. These cell lines are under further characterization by sequencing for mutations in known human oncogenes.
Interestingly, this resource can be screened on demand for any chemical or biological agent. As an example, the NCI60 has been recently used to determine the permissivity of standard cancer cell lines to VACV infection and replication, with the aim to better characterize viral oncolytic therapeutic strategies [ 54 ].
The emergence of tumor acquired resistance to pharmacological inhibitors linked to mutations in driver oncogenes has recently revived the interest for cancer cell lines. Indeed, an extensive characterization of cell lines at the genomic and genetic levels will allow determining a genetic profile predictive of drug sensitivity. Such a signature will help to stratify patient population and identify efficient therapeutics combination, as long as cell lines reflect real tumor biology.
Patient-derived xenograft models for oncology drug discovery
Using current high throughput techniques this program intends to provide information on mutations, copy number variations, single nucleotide polymorphisms SNPs and microsatellite instability of usual cancer cell lines. On the other hand, 5 out of the RNAi-targeted genes had an antagonistic effect on MTS in all four cell lines in the screen, and eleven out of the RNAi-targeted genes had an antagonistic effect on MTS in at least two cell lines. Validation studies were able to confirm modifier effects for four genes. The study also strongly demonstrates that a panel of heterogenous cell lines needs to be included in these types of assays, as results can be diametral from one cell line to another.
The LO is more or less identical with the classical drug development process. The process will be adapted on the validated targets and includes assay and model development, followed by a screening phase of selected compound, peptide, antibody, or RNAi libraries to identify a lead structure Figure 1. Once a lead structure has been identified, optimization processes are started, frequently in parallel for several leads.
As the most difficult part of the targeted drug development, this part can be seen as an extended lead and target discovery phase, addressing the molecular mechanism of action in correlation to optimal pharmacodynamic activity physiological mechanism of action , optimal pharmacokinetics PK [absorption-distribution-metabolism-excretion ADME ], toxicity, as well as resistance development. A large number of functions are now involved in this integrated preclinical drug development IPDD, Figure 5 , including functions like medicinal and protein chemistry, cell and structural biology, pharmacology, PK and early toxicology Tox.
Data from the screening, now implemented in large data bases, will be further used for computational modelling. A broad panel of lead optimization tasks and criteria for oncology drug development has been established, which should address:. Similar to the TIV process, increased demands on the lead optimization have changed the requests on the disease models. The target driven approaches now require models with defined levels of target expression which will be mainly generated by genetic modifications and cloning:.
For example, several studies, performed during the development of the already mentioned new MTS, will be discussed.
Mechanistic Models in Oncology
To overcome limitations associated with the established drugs, compounds from different structural classes have been synthesized and tested for activity 6. Extensive preclinical in vitro studies have been set up to demonstrate improved target activity for these new compounds 7. A defined panel of tumor cell lines sensitive and multi-drug resistant was tested in comparison to the available standard paclitaxel and found to be strongly sensitive to the new MTS with only moderately variations in response IC50 between 0. So far, no natural resistant cell line was identified and even treatment for more than one year with the new MTS did not result in development of resistance unpublished own results.
Further mechanistical investigations in tumor cell lines demonstrated, that the new MTS induces a more rapid and potent tubulin polymerization than paclitaxel. A rapid and effective influx into cells, combined with the evasion of P-glycoprotein efflux pumps, have been identified as key qualities resulting in consistently more potent activity than microtubule-stabilizing taxanes 8. However, in line with other MTSs, it causes mitotic arrest, followed by activation of the mitochondrial apoptotic pathway.
Profiling of the pro-apoptotic signal transduction pathway using a panel of small interfering RNAs revealed that it acts in a fashion comparable to paclitaxel. Further mechanistic studies in lung cancer cells 9 revealed a concentration-dependent disturbance of cellular organization with two apparent phenotypes.
Interestingly, the treatment with low doses effectively inhibited cell proliferation, but—compared to high concentrations—induced apoptosis only marginally. Analysis of differential gene expression in tumor cells treated either with high and low drug concentration demonstrated a non-overlapping set of regulated genes:.
Models of Models: A Translational Route for Cancer Treatment and Drug Development
In contrast, treatment with the low concentration revealed an up-regulation of direct transcriptional target genes of TP53, like cdkn1a , mdm2 , gadd45a and fas. This resembles an activation pattern which is caused in response to mild, repairable damage, and induces cell cycle arrest, rather than strong damages which promote apoptosis.
This allows repair processes to take place and the cells to survive. Knockdown of TP53 led to a significant increase in apoptosis induction 9. These mechanistic data confirmed, that up-regulation of TP53 and its downstream effectors by low concentrations of MTS is responsible for the relative apoptosis resistance of A lung cancer cells and might represent a new mechanism of resistance Figure 7.
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A different phenotype appears to be induced at higher MTS concentrations, with progressively more perturbed microtubule dynamics, formation of microtubule bundles and activation of the SAC leading to an arrest in mitosis. Mainly, this result in an induction of mitochondrial apoptosis, mediated by members of the Bcl-2 family proteins, and is substantially similar to that seen with paclitaxel and other epothilones 8.
But, mitotically arrested cells may also undergo aberrant mitosis or mitotic slippage and endo-reduplication. The variations in the extent of apoptosis among breast cancer cells after MTS treatment could be explained by differences in the apoptotic signalling rather than by differences in mitotic arrest. The U. Requirements set in this document make clear, that the qualification process for a biomarker has many parallels to drug discovery and development, starting with biomarker identification and validation, followed by assay development and optimization, and finally followed by validation in clinical trials.
In the preclinical oncology research departments from most pharmaceutical and biotech companies, the TR has now become an integrative part of the development. Considering the heterogeneity of cancer, it has become clear that this research requires new approaches. During the development of our previously mentioned MTS, we have addressed the questions for a predictive biomarker in lung cancer patients with a new type of preclinical study.
This was based on the observation, that interestingly, some tumor models, i. Human tumors accumulate genetic and molecular abnormalities, leading to broad heterogeneity. Large panels of tumor models reflecting tumor heterogeneity might have increased value for predicting the response to new therapeutic agents in the clinic. Consequently, it is important to use a large panel of clinically relevant tumor models for translational studies. However, from the in vitro studies with 20 breast cancer cell lines and in more than 30 other cell lines, we have not been able to identify natural resistance mechanisms to MTS.
This led us to work with extended panels of in vivo models. To address this discrepancy between in vitro and in vivo activity, further studies across a panel of human lung cancer xenograft models were performed Figure 8A. In this heterogeneous panel response to MTS—treatment was determined in an integrative preclinical phase II design—further resistant tumors were identified Figure 8B. Tumors with wild-type TP53 as well as high expression of genes involved in cell adhesion, hypoxia or angiogenesis were more likely to be resistant to MTS treatment For validation, combination experiments were performed with drugs or siRNA is, targeting some of the identified resistance mechanisms, i.
Indeed, when combined with MTS treatment, combination therapy resulted in restored anti-tumor activity in resistant tumor models [ 9 , 11 , unpublished own results ]. Hypoxia triggers pathways that drive angiogenesis and tumor progression, and the presence of genes associated with these pathways has previously been associated with a negative prognosis and resistance to therapy Up-regulation of CA9 and CA12 gene expression, in particular, has been detected in a large number of common malignancies and is implicated in tumor development In cell culture studies, we performed earlier, treatment of A cells with low concentrations of MTS resulted in stabilization of TP53 and induction of TP53 target genes, potentially resulting from consistent translation of the long-lived TP53 mRNA during prolonged mitosis TP53 check point induction by low MTS concentrations targets genes such as cdkn1a or gadd45a and induces cell cycle G1 arrest, rather than promoting apoptosis 15 -