Biochemical Pharmacology 89 (2014) 313–320

Commentary

In vivo models of multiple myeloma (MM)

Eric Sanchez, Haiming Chen, James R. Berenson *

Institute for Myeloma & Bone Cancer Research, West Hollywood, California, USA

A R T I C L E I N F O

Article history:

Received 30 January 2014

Accepted 25 March 2014

Available online 1 April 2014

Keywords:

In vivo

Multiple myeloma

Pre-clinical

Xenografts

Severe combined immunodeficiency (SCID)

mice

New chemical entity (NCE)

A B S T R A C T

The development of the plasma cell tumor (PCT) model was the first widely accepted in vivo model of

multiple myeloma (MM). Potter and colleagues used this chemically induced PCT model to study the

pathophysiology of malignant plasma cells and also used it to screen anti-MM agents. Two decades later

the C57BL/KaLwRij mouse strain was found to spontaneously develop MM. Testing of pamidronate using

this endogenously arising MM model revealed significant reductions in MM-associated bone disease,

which was subsequently confirmed in human trials in MM patients. Transgenic models have also been

developed in which the MM is localized in the bone marrow causing lytic bone lesions. Experiments in a

transgenic model showed that a new oral proteasome inhibitor was effective at reducing MM burden. A

clinical trial later confirmed this observation and validated the model. The xenograft model has been

used to grow human MM in immunocompromised mice. The xenograft models of MM have been very

useful in optimizing drug schedules and doses, which have helped in the treatments given to MM

patients. However, in vivo models have been criticized for having a low clinical predictive power of new

chemical entities (NCEs). Despite this, the knowledge gained from in vivo models of MM has without a

doubt benefited MM patients.

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Biochemical Pharmacology

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1. Introduction

In vivo models have historically been used to evaluate newtherapies and targets for the treatment of cancers. First, we discussthe historical development of heterotransplantation. Next, wedescribe how mouse multiple myeloma (MM), transgenic MM andhuman xenograft models have been used to evaluate the efficacyof new chemical entities (NCEs), and whether clinical studies havebeen performed which confirm their use for predicting respon-siveness to NCEs. Given that MM is known to be a very difficultcancer to grow in vitro and in vivo; and, thus, a lack ofheterogeneous MM xenograft models currently exists, the historyof whether MM xenografts will be better predictors of NCEs thanxenograft models of various other cancers is yet to be written.However, although more MM xenograft models are clearly neededbefore conclusions can be drawn, optimization of dosing andscheduling of anti-MM therapies has come directly fromthe current/existing MM xenograft models. Conversely, NCEswhich were first tested in MM patients have subsequently beentested in mouse, transgenic and human MM xenograft models.

* Corresponding author. Tel.: +1 310 623 1214; fax: +1 310 623 1120.

E-mail addresses: esanchez@imbcr.org (E. Sanchez), hchen@imbcr.org (H. Chen),

jberenson@imbcr.org, tgarceau@imbcr.org (J.R. Berenson).

http://dx.doi.org/10.1016/j.bcp.2014.03.013

0006-2952/� 2014 Elsevier Inc. All rights reserved.

These models have been validated by being responsive to NCEsadministered to humans. The objective of this commentary is toprovide readers examples of various in vivo models of MM, frompast to present, how they have been used to evaluate the efficacy ofNCEs and/or have been subsequently validated, provides reasonswhy the attrition rate of NCEs entering clinical trials is high andwhat can be done to lower the attrition rate.

1.1. Heterotransplantation

Animal models of cancer have long ago been of scientificinterest. In 1912, James B. Murphy demonstrated that a ratsarcoma was capable of growing in the outer membrane (fusedchorion and allantois) of chick embryos [1]. It could grow fromembryo to embryo and could also be implanted back into the ratafter generations of growth in the chick embryo. He demonstratedthat small round lymphocytes, derived from adult chicken spleenand bone marrow, were responsible for the rejection of thesarcoma [2]. Dr. Murphy demonstrated that cells of at least twoorgans (spleen and bone marrow) were immunologically compe-tent and able to reject tissue grafts. Heterologous transplantationwas demonstrated a few decades later by successful growth andserial transfer of rabbit tumors into the anterior chamber of the eyeof guinea pigs [3]. This experiment suggested that it might bepossible to grow human cancers in mammals. In 1938, Harry S.N.

E. Sanchez et al. / Biochemical Pharmacology 89 (2014) 313–320314

Green briefly mentioned the growth of a human scirrhus cancer of thebreast using this inoculation method [4]. In 1950, successful hetero-transplantation of human melanomas into the anterior chamber ofthe eye of guinea pigs was demonstrated [5]. Detailed literaturedealing with heterologous transplantation of mammalian tumors isavailable by each of these and other authors, as other claims to firstsuccessfully grow human cancers in foreign species have been madebut were not substantiated upon critical scientific review.

In 1960, experiments demonstrated that human cell lines couldgrow in rats if injected less than 24 h after birth [6] and otherinvestigators confirmed this observation [[7,8], respectively]. Therole of the thymus was unknown in the early 1960s. Prevailingopinion at the time was that the thymus was a graveyard for dyingcells. In 1962, J.F.A.P. Miller made a major discovery inimmunology when he demonstrated that mice thymectomizedat birth failed to reject skin from a different species, the rat [9].The marked deficiency of lymphocytes which was accompanied byan inordinate susceptibility to infection in neonatally thymecto-mized mice provided the rationale for grafting mice with foreigntissues. The mouse mutant ‘‘nude’’ mouse was first described in1966 [10] and soon thereafter it was found not to contain a thymus[11]. The marked deficiency of T lymphocytes in nude miceprovided the rationale for grafting mice with human tissues.This discovery was a major scientific breakthrough as this immunedeficient mouse provided scientists a model which allowed studiesof the biology of human cancer in vivo and therapies directedagainst it without placing patients at risk. Less than two decadeslater, severe combined immunodeficiency (SCID) mice weredescribed [12], also allowing for the growth of human cancersin them. Collectively, these mouse strains (nude, SCID) led to thecreation of the human xenograft model (discussed in Section 3),which have allowed large-scale testing of NCEs against humantumors and is the modern animal model system in use today.

2. Mouse MM Models

2.1. BALB/c Models

In the late 1920s and 1930s, observations were made whichdemonstrated the carcinogenicity of hydrocarbons administeredto immune competent mice [13,14]. In 1950, it was shown thattumors were induced in susceptible mouse tissues by the useof various carcinogenic hydrocarbons [15,16]. One hydrocarbon (oroil) in particular caused two cases of plasma-cell leukemia [15].Peritoneal plasma cell tumors (PCT) were discovered in 1959 whenplastic objects were implanted intraperitoneally into BALB/c mice[17]. Subsequently, the most common oil used to induceplasmacytomas in the peritoneum of mice was pristane [18].These chemically induced tumors secrete monoclonal immuno-globulin (Ig), when housed under conventional conditions [18].However, there are differences when comparing these chemicallyinduced murine plasmacytomas (MPCs) to human MM. It wasshown that most human MM secrete IgG whereas most MPCssecrete IgA [19]. One disadvantage of this model was that theplasma cells were localized to the peritoneum and subcutaneousspace, but not in the bone marrow. However, in the early 1960s thiswas the best available model used to study MM. It provided aninsight into the pathophysiology of MM. Currently, one benefitof this mouse PCT model is that the BALB/c mice are immunecompetent, so it is possible to study the interactions between thePCT and immune system. In certain experiments where theimmune system is needed, this model is of great value. Forexample, it has been established that one of the mechanisms ofaction of immunomodulatory drugs (thalidomide, lenalidomideand pomalidomide) for the treatment of MM patients is byenhancing the immune system to fight the cancer [20]. Had

immunomodulatory drugs been available at the time of thecreation of the PCT model (1959), the PCT model would havepredicted clinical efficacy of these agents for the treatment of MMpatients; and, thus, improving the prediction rate of animal modelstested using NCEs. The PCT model would have also predictedclinical efficacy of bisphosphonates, like zoledronic acid, had thesedrugs existed in the early 1960s. In 2005, a study was publishedwhich evaluated the effects of zoledronic acid in these chemicallyinduced mouse PCTs. The results demonstrated a significant delayin PCT development in the group receiving zoledronic acid pluspristane when compared to the group receiving only pristane orthe group which received pristane plus zoledronic acid after theappearance of PCTs [21]. It is now well known that zoledronic acidis effective for the treatment of MM patients [22–26] and a recentstudy has clearly demonstrated a survival advantage for MMpatients receiving this potent bisphosphonate [27].

2.2. 5T MM Models

In 1985, it was discovered that aging mice of the C57BL/KaLwRijstrain spontaneously develop MM [28]. Many sublines were createdand denoted as 5T MM. For example, a cell line from this model wasestablished and called 5T33 [29]. It was shown that some, but not all,aged mice predominately secrete excessive IgG in the serum andbone marrow analysis revealed myeloma cells which produce theosteolytic lesions, both of which occur in human MM. Preclinically,the 5T MM model was used to test the effect of pamidronate (abisphosphonate) for the treatment of MM bone disease, wherereductions in MM-associated bone disease were observed in mice[30,31]. These promising animal studies provided the rationale toconduct trials to test the efficacy of bisphosphonates (i.e., pami-dronate and zoledronic acid) for MM patients. Berenson et al.published the results of a double-blind, placebo-controlled random-ized trial involving 392 patients with stage 3 MM, who receivedpamidronate. Significant reductions in skeletal events and reductionin pain were observed among pamidronate-treated patientscompared to those patients receiving placebo [32]. A survivaladvantage in a subgroup of MM patients who were on salvagetherapy who were treated with pamidronate was also demonstrated[33]. These studies provide an example of how the testing of NCEs onMM animal models have successfully predicted clinical efficacy forpatients with MM. Conversely, clinical studies have validated the 5TMM model as a good model of human myeloma. The first therapeutictrials evaluating zoledronic acid were conducted in 1996. Berensonet al. conducted a study evaluating the effects of zoledronic acid (amore potent bisphosphonate than pamidronate) for MM and breastcancer patients which showed a reduction in several bone resorptionserum markers and improvement in bone density [22]. Body et al.also conducted a Phase I trial, which demonstrated that administra-tion of zoledronic acid resulted in normalization of serum calciumlevels in patients with tumor-induced hypercalcemia [23].The preliminary human data obtained by these authors whichindicated that intravenous infusions of zoledronic acid was safe,inhibited bone resorption and decreased bone pain was subsequentlyconfirmed in larger trials [24–26]. At the time of these latter clinicaltrials, the 5T2MM model was also used to test effects of zoledronicacid in mice. The animal data showed that it was able to prevent thedevelopment of osteolytic bone disease, decrease tumor burden inthe bone marrow, and increase survival [34,35]. Thus, MM animalmodels can predict efficacy of NCEs advancing into clinical trials, andclinical studies can, in turn, validate the use of the models.

2.3. Transgenic MM models

Many transgenic MM models (or genetically engineered mousemodels, GEMMs) have recently been developed. The first type of

E. Sanchez et al. / Biochemical Pharmacology 89 (2014) 313–320 315

transgenic MM models was in a single-transgenic mouse [36–39].In addition to the plasmacytomas that arise in these models, theyhave also been shown to develop lymphomas in some cases [37–39].In the clinical setting, the coexistence of MM with lymphomasoccurs only very rarely. Furthermore, the plasmacytomas intransgenic MM models were not tested with anti-MM agentsknown to be clinically effective in MM; and, thus, they have not beenthoroughly validated as MM models. In an effort to accelerate tumoronset and increase tumor incidence, double-transgenic mice havebeen developed in which tumor onset occurs more rapidly and in ahigher proportion of the mice. Double-transgenic Myc/Bcl-XL micedevelop bone marrow plasma cell tumors which secrete monoclonalIg and cause osteolytic lesions [40]. A disseminated model (DP54-Luc) was developed from this double-transgenic Myc/Bcl-XL modelby culturing the cells in vitro [41]. These cells were then injected intoNOD (non-obese diabetic)-SCID mice, resulting in rapid death due todiffuse growth of MM plasma cells in various organs in these mice.Treatment with MLN9708 was evaluated in both MM models andsignificant anti-MM activity was observed [41]. Subsequently, aclinical trial demonstrated the efficacy of this new anti-MM agent inMM patients [42]. Activation-induced deaminase (AID)-dependentactivation of MYC in germinal center B-cells of VkMYC mice resultedin the progression of benign monoclonal gammopathy to an indolentMM with biological and clinical features characteristic of humanMM, resulting in the creation of a new MM model [36]. The tumors inthese mice mostly secrete monoclonal IgG. These mice are anemicand have plasma cells that are localized to the bone marrow. Somemice were found to contain bone lesions. Vertebral collapse has alsobeen observed and decreased bone mineral density has beenoccasionally detected. Significant reductions in IgG were observed inmice treated with conventional therapies for MM [36,43]. However,romidepsin showed significant activity in this MM model [43], yet inthe treatment of MM patients it has been abandoned due to a lack ofsignificant clinical activity when administered alone or in combina-tion with conventional anti-MM agents. Conversely, mice treatedwith agents that have not shown much anti-MM activity in humansalso did not show significant reductions in IgG [36,43]. Whenassessing the model to predict the efficacy of a NCE (SNS-302) in MM,it failed, as it showed efficacy at reducing IgG (a marker of tumorburden) in mice whereas a clinical study resulted in early studyclosure and concluded that SNS-302 had limited clinical activity inheavily pretreated patients with MM [44]. Whether or not thismodel will pass or fail in predicting the efficacy of other NCEs iscurrently unknown as clinical trials evaluating these agents in MMhave not been reported. If clinical trials for MM patients areconducted, we will have a better idea as to whether or not this modelis truly predictive of NCEs. A very small number of lymphomas havebeen observed in this model [45], in contrast to other transgenicmodels [37–39]. Another transgenic model is of a MGUS/MMphenotype in mice with Em-directed expression of the XBP-1 splicedisoform (XBP-1s) [46]. In human MM, the transcription factor XBP-1is a major regulator of the unfolded protein response (UPR) andabundant expression of XBP-1s has been detected in MM cells [47].MM cells are heavily reliant on the ER for IgG synthesis as thefunction of the UPR is to fold unfolded proteins and correctly foldmisfolded proteins which accumulate under ER stress conditions.Em-xbp-1s transgenic mice develop elevated serum IgG, suben-dothelial Ig deposition and bone lytic lesions. Collectively, thesetransgenic mouse MM models are an additional tool which will helpus gain a better understanding and find better treatment options forMM patients.

3. Human MM xenografts

Although xenograft models which allowed the growth ofhuman cancers in athymic mice were developed in the late 1960s

and early 1970s [48], human MM xenografts have been difficult toestablish in athymic mice [49]. In 1983, the SCID mutation wasfound to occur spontaneously in mice of the BALB/c C.B-17 strain,which were found to have low levels of B and T cells [12].The successful engraftment of primary human MM cells in theperitoneum of SCID mice was demonstrated in 1992 [50]. HumanMM cell line growth using human fetal bone implants wasdemonstrated in 1997 [51]. Irradiated SCID mice were implantedwith two human fetal bone grafts and human MM cell lines wereinjected only into the marrow cavity of the left bone implant. MMcell lines engrafted and proliferated first in the left and then in theright human fetal bone implant. However, the human MM cell lineswere also found in many other organs in the SCID mice that did nothave the bone implants. The growth of primary MM cells inirradiated SCID mice implanted with human fetal bone wasdemonstrated a year later, and it was observed that primary MMcells did not grow in the murine bone marrow and blood, but onlygrew in the human fetal bone [52]. While these models were usedto study the myeloma and its manifestations, their usefulness islimited. For example, models which use primary MM cells are notuseful as chemotherapy screening systems in vivo due to theinability to generate large cohorts of animals bearing tumors.The MM models which use MM cell lines may not be good models,as MM cell lines are developed from the most advanced cases ofextramedullary myeloma, which is not representative of themyeloma seen from most patients in the clinic and are maintainedalone ex vivo for many years prior to their implantation. It is wellrecognized that the microenvironment is a critical part of thepathogenesis of myeloma.

During the past decade, we (Institute for Myeloma and BoneCancer Research) have successfully grown and maintained tumorsderived from MM bone marrow or blood from plasma cell leukemia(PCL) patients in severe combined immunodeficient SCID mice [53].The models display many features, both morphologic and immuno-logic, of human MM and PCL that are especially suited to preclinicaldrug studies. Initially, SCID mice are surgically implanted with a 20–40 mm3 bone marrow biopsy sample, from a MM patient, into theleft hind limb. From the growth and passage of the implantedsample, we have established ectopic MM models. The morphology ofthe MM tumors growing in SCID mice remains unchanged and isidentical to that from the human tumor. There is nearly a 100%success rate at passing these tumors from mouse to mouse, and wealso are able to grow MM tumors in a short period of time. Humanmonoclonal immunoglobulin (hIg) levels are detected by ELISAafter 7 days and a visible tumor as soon as after 14 days post-implantation. Notably, the growth pattern of the tumor and therise in hIg levels is very similar among different SCID mice bearingthis tumor. The following figure outlines the procedures used togenerate human xenografts of MM (Fig. 1).

For serial passage of the tumors, mice are sacrificed and MM cutinto small tumor fragments and intramuscularly implanted intorecipient mice. These tumors have been maintained for the pastdecade by serial passage. The growth rate of each MM tumor variesbut the average time to passage into another host is approximately6 weeks. With these MM models, we are able to quickly generatelarge numbers of MM-bearing animals for pre-clinical studies.These solid MM tumors can also be injected subcutaneously orintravenously (i.v.) [53]. If injected i.v., some mice develop hindlimb paralysis, suggesting spinal cord compression. However, i.v.injection of human MM tumor lines and MM cell lines intounconditioned and irradiated SCID mice results in a disseminatedMM model [[54] and [51], respectively, where the injected cellsgrow and form solid MM tumors at many different anatomicallocations including the stomach and subcutaneous space. They donot specifically home to and exclusively grow in the bone marrowof SCID mice. It has been shown that primary human MM cells only

Fig. 1. A 20–40 mm3 bone marrow biopsy sample from a MM patient is surgically implanted into the left hind limb of a SCID mouse. The use of the biopsy allows implantation

of both the malignant population and all of the supporting cells and structures in the BM from the MM patient. This overcomes the disadvantages of using cell lines or fresh BM

aspirates which lack these additional elements which are critical to the establishment and growth of human MM in the mouse.

E. Sanchez et al. / Biochemical Pharmacology 89 (2014) 313–320316

grow in the human microenvironment [52]. In fact, based on theliterature, a MM xenograft model where the injected primaryhuman MM cells specifically home to and grow in the bone marrowof normal or immune compromised mice does not exist [[52,55],our unpublished observations]. Such a model would be of greatvalue as the only MM cells which exclusively grow in the bonemarrow of mice are of mouse origin (C57BL/KaLwRij mouse MMmodel and GEMMs). Despite this, the ectopic models (intramus-cularly growing, solid MM tumors) we have generated at ourinstitute are specifically designed to evaluate the anti-MM activityof NCEs alone and in combination regimens with conventionaltherapies. They have proven highly useful to many pharmaceuticaland biotech companies, ranging in size from small to large, for theclinical development of different anti-MM treatment strategies.The dosing schedule of pegylated liposomal doxorubicin (PLD) weuse in our clinic is based on data generated from our pre-clinicalanimal models. Using our MM xenograft model, we showed thatadministration of low-dose PLD daily markedly improved anti-MMeffects and was better tolerated than PLD given at a higher doseonce weekly [54]. Our pre-clinical results suggested that lowerdoses of PLD administered more frequently would improve itsefficacy as well as reduce its toxicity. Thus, we decided todetermine if this would translate into the clinic. We evaluated 28patients with relapsed/refractory MM who received lower butmore frequent doses of PLD, bortezomib and dexamethasone [56].The use of this modified, ‘‘metronomic’’ dosing regimen demon-strated that it was just as effective, and better tolerated thanregimens using these same three agents at higher doses and differentschedules [56–58]. Furthermore, we also evaluated this lower butmore frequent dosing of PLD, bortezomib and dexamethasone in thefrontline setting for MM patients and it showed improved tolerabilityand safety while maintaining a high response rate when compared tostandard treatment with these same agents [59].

MM cells from the in vivo MM models we have developed do notgrow indefinitely in vitro. This is in contrast to MM cell lines whichgrow in vitro, and similar to primary MM patient cells which do not.These MM tumors can only be maintained via serial in vivo passagein SCID mice. In vitro culture of MM cells from our in vivo models,however, does allow a window of opportunity to test the directanti-MM effects of anti-cancer agents as they can survive in culturefor a week. This provides us the opportunity to evaluate the directanti-MM effects of NCEs and the corresponding mechanism ofaction. An advantage of in vitro methods is that the cost associatedwith these experiments is substantially less expensive than in vivo

experiments. The disadvantage is that since anti-cancer drugs willbe given to humans, in vitro cancer models do not allow for theevaluation of the potential toxic effects of NCEs on organs andorgan systems. Another difference between our in vivo MM modelsand traditional MM cell lines injected into SCID mice is that ourtumors secrete IgG (similar to MM patients), whereas in most MMcell lines this has been lost. Comparisons of the pre-clinical toclinical outcomes of drug responses between conventional MM celllines, most of which do not secrete IgG, and MM tumor lines likeour own, which do secrete monoclonal protein, have not beendetermined. However, comparisons between the pre-clinicalmodels can be made. Based on the published literature we haveobserved that our MM xenograft models, when compared to MMcells lines growing in SCID mice, tend to be more resistant to anti-MM therapies. Whether in vivo MM models which secrete IgG arebetter predictors of drug responses in humans, when compared toMM cell lines grown in animal models, is currently unknown.

4. Conclusions and perspectives

Recently, there have been major advances in the treatment ofMM. Among these, our in vivo models of MM have led to clinical

E. Sanchez et al. / Biochemical Pharmacology 89 (2014) 313–320 317

trials which have directly benefited patients [56–59]. Theexamples mentioned in this commentary are those in which in

vivo models of MM have successfully predicted the clinical efficacyof NCEs; however, as a whole, in vivo model systems have failedand have been criticized for having a low clinical predictive power[60,61]. Thus, the question as to why animal models continue todictate how NCEs are advanced to the clinic has been raised.However, for several reasons, we believe that the failure of MManimal models to predict NCE efficacy in humans is not due to theanimal models per se. One of our concerns is the lack ofheterogeneity of the existing MM xenografts, as these modelsare extremely difficult to establish. Presently, what typically occursis that a NCE is tested against a couple of MM xenograft models,and if activity is observed in one, a manuscript is written statingthat the NCE is active against MM and that a clinical trial iswarranted. Instead, what is needed is the development of a variety,or a ‘‘library,’’ of genetically distinct MM xenograft models, forexample 100, and each unique tumor tested against a single NCE.We believe that with more MM xenograft models scientists couldmore accurately predict whether clinical efficacy of an NCE wouldbe observed, as it is well known that in MM patients there is greatgenetic heterogeneity resulting in variable responses to differenttherapies [45]. Currently, however, this is not the case as nobodyhas figured out how to increase the engraftment rate of human MMtumors growing in immune compromised mice. We believe thatthis is the main reason for the failure of existing MM xenograftmodels to predict the clinical efficacy of NCEs. Although eachpatient has the same cancer, MM, each is genetically different anduniquely responsive to different combinations of drugs over thecourse of their disease. Similarly, a NCE which works in one MManimal model may not work in other MM animal models, and nosingle animal model will ever be an absolute predictor of thehuman response. Thus, significantly more MM animal models areneeded in order for us to be able to accurately predict clinicalefficacy of NCEs.

The discovery of the nude mouse allowed for the first successfulgraft of a human cancer in 1969 [48]. However, it has been shownthat certain types of cancers are more prone to grow in this mousestrain while others are not. Mattern et al. reported a 32% take ratewhen human lung tumors were implanted into nude mice [62].Fogh et al. reported a 47% and 54% take rates in nude miceimplanted with lung and colon carcinomas, respectively [63].However, breast cancers are more difficult to grow in nude mice.Giovanella et al. implanted 433 breast carcinomas obtained frompatients and 28 grew in nude mice, a 6% successful transplantationrate or ‘‘take rate’’ [64]. Similarly, Fogh et al. reported a 9% take ratefor human breast carcinomas [63]. As for MM, it is well known thathuman MM xenografts are difficult to establish in nude mice [49],and the authors are not aware of any reports in the literaturedemonstrating the establishment of a MM xenograft model in thismouse strain from a primary MM sample.

As mentioned in Section 3, MM xenografts have been generatedin CB17 SCID mice [52,53]. However, the take rate is low (<10%).While certain types of MM xenograft models are used to study MMand its manifestations, they are limited to the amount of tumorcells obtained from each patient, and are not useful as NCEscreening systems in vivo due to the inability to generate largecohorts of animals bearing MM tumors [50–52]. Furthermore,primary MM cells die after complete resorption of the fetal boneimplants. Solid MM tumors (ectopic MM models) are needed fordrug studies as they need to be implanted either intramuscularlyas we do in our studies [54] or subcutaneously. Our ectopic MMmodels allow us to measure the anti-MM effects of NCEs usingtumor volume mass because the MM tumors do not spread to otherorgans within the mouse (we can also measure human IgG levels inplasma). Both parameters of tumor growth are evaluated when

assessing the effects of NCEs. It is well known that most primaryMM cells from patients do not grow solid MM tumors in SCID mice.Thus, the frequent development of ectopic MM xenograft modelswould be a major scientific breakthrough as it would facilitateidentification of NCEs, saving pharmaceutical companies millionsof dollars by avoiding costly clinical trials, and may allow forpersonalized therapy for MM patients if their individual tumorscould be consistently grown.

Given the low take rates when human primary MM samples areimplanted into SCID mice and the necessity for more MM xenograftmodels, it is our goal to develop additional xenograft models ofhuman MM. In attempt to grow more models with higher takerates, freshly obtained bone marrow cells from MM patients havebeen mixed with pristane and implanted into mice. The rationalefor this experiment was based on Potter’s experiments where Balb/c mice were injected with pristane and after approximately oneyear plasma cell tumors developed [18]. One of the main growthfactors responsible for the growth of these PCTs was IL-6 [65,66],which was shown to be produced by macrophages. Since SCID micehave functional macrophages, it was hoped that the primary MMcells and MM biopsies would grow. This same experiment was alsoconducted but the human IL-6 was injected along with the primarycells mixed with a gel matrix (to contain the cells and cytokine in alocalized area, preventing diffusion). Further immunosuppressionof SCID mice by administration of cyclophosphamide alone,cyclosphorine A alone, and a combination of both immunosup-pressive drugs have been injected in SCID and nude miceimplanted with primary MM cells and MM biopsies alone andin combination with IL-6 (our unpublished experiments).The rationale for using cyclosporine A in SCID mice lackingfunctional T cells is because of the possibility of residual human Tcells in the bone marrow aspirate from contaminating peripheralblood and the bone marrow biopsy itself. The purchasing ofdifferent immunodeficient mouse strains has also been evaluated(i.e., nude mice), to see if perhaps the lack of human MM growth inthe SCID mice we use is due to the mouse strain itself.Unfortunately, none of the above mentioned experiments hasyielded solid MM tumors in SCID mice. Review of the literature onindividuals who have implanted primary MM cells and solid MMtumors grown in immunocompromised mice is lacking. Further-more, companies and laboratories which specialize in developingpatient derived tumors (a.k.a. xenograft) report success withcancers such as colon, lung, breast and prostate, but not MM. Thiscancer is known to be especially difficult to grow, both in vivo andin vitro. There must be additional factors beyond immunosuppres-sion, which prevents human MM growth in immunodeficient mice.

A second factor contributing to the low predictive efficacy ofMM xenograft modes is how little funding is available forpreclinical animal research. Assuming that more monies becomeavailable, what will be needed is for pharmaceutical and biotechcompanies to invest more money preclinically to test a single NCEusing many different MM xenograft models, before rushing intoexpensive clinical trials. Currently, only a small portion of fundingis available for preclinical NCE studies and as soon as a signal isobserved in an animal model of MM, millions of dollars are spent on aclinical study. Once more MM xenograft tumors are availablepreclinically, a shift needs to be made to spend more money onpreclinical studies so that we are able to predict with higher accuracythan we currently do whether a NCE will show clinical activity.

Perhaps the creation of a ‘‘library’’ of MM xenograft tumors maystill not be representative of most MM seen in the clinic; and, thus,may not be useful as a screening system to test NCEs. If this laterscenario turns out to be true, then the future of MM xenograftmodels in assessing the therapeutic utility of NCEs will have to bepatient specific. Specifically, personalized xenografts where thepatient’s tumor is grown in mice and NCEs are evaluated against

Table 1Timeline: the development of in vivo models of MM.

Year 1959 1985 1992–present 2002–present

MM animal model PCT 5T MM Human MM xenografts Transgenic MM

E. Sanchez et al. / Biochemical Pharmacology 89 (2014) 313–320318

only their specific tumor. Compared to having a panel of MMxenograft models to test NCEs, this will be cumbersome. However,this may very well turn out to be the case. A bone marrow biopsy oraspirate may have to be obtained from each MM patient and theirspecific tumor grown in a mouse. Once their tumor has grown itwould then be implanted in many more mice and NECs (andconventional therapies) tested against that patient’s unique tumor,to determine what drug combinations work for that specificpatient. An example of this type of scenario has proven successfulfor patients with pancreatic cancer. Xenograft models weredeveloped from patients with this type of solid tumor, and thepreclinical data used to guide specific treatment for each patient.Eighty-eight percent of the treatment plans which worked in thepersonalized xenografts were effective (defined as durable partialremissions) in the corresponding human tumor [67]. If this form ofpersonalized medicine for patients with cancer is, in fact, theoptimal model system of choice for MM patients, then a list ofeffective treatments for the patient would be preclinicallygenerated and provided to the physician. However, there couldbe selection bias that would decrease the efficacy of thesepersonalized MM xenograft models to predict clinical efficacy ofNCEs and their combination therapies. For example, what if aphysician chooses an effective, but not the best, drug combinationfrom the list because only certain drugs are fully reimbursed byinsurance companies? The best drug combination for the MMpatient may not be tested, thus lowering the predictive efficacy ofthe MM xenograft model against NCEs and combination therapies.A related concern is that patient specific xenograft models may notbe practical for most MM patients. For patients who do not havethe financial means, who is going to cover the costs to purchasehundreds of immune compromised mice, house them in a sterileenvironment, purchase drugs to be tested, and the animaltechnician administering the NCEs and collecting, graphing andanalyzing the data/results?

We, scientists, also have to master how to interpret the animaldata we obtain in order for the successful translation into the clinic.Differences between how tumors grow in humans and mice haveto be taken into consideration. For example, in addition to thedifferential slower growth rate of tumors in man than theirxenografts in mice and a faster metabolism in mice [62], blood flowvia increased angiogenesis has been observed to contribute to thegrowth rate of human tumor xenografts [68]. These authorsreference an article which showed that poorly perfused tumorshad a decreased response to radiation and altered sensitivitytoward anti-proliferative drugs. They recommend that blood flow-related data should supplement conventional factors (histologicalclassification, clinical staging and grading) in order to bestindividualize therapy.

Chemoresistance is a significant problem among MM patientsand all patients will progress from each of their treatments. Thus,they will require many different anti-MM regimens during theircourse of disease. In addition to being able to predict moreaccurately the clinical efficacy of NCEs, the generation of more MMxenograft models will enable scientists to grow a patient’s tumorbefore and after drug resistance. A patient’s tumor could be grownin immunocompromised mice, testing of several anti-MM thera-pies (and NCEs) performed and effective ones determined. Thepatient would then be given only the treatment regimen(s) whichworked on their tumor in mice. As chemoresistance to the effectivetherapy will eventually develop in the patient, their drug resistantMM tumor could then be obtained again and grown a second timein more mice. Scientists could then test their drug resistant MMtumor by screening additional NCEs alone and in combination withconventional MM therapies. Again, only effective drug combina-tions which work in mice would be given to that patient. In theory,this process could be repeated indefinitely and help clinicians

decide which therapies will likely be effective following a drugresistance in a specific MM patient’s tumor.

Increasing the predictive value of the existing MM models, andlowering the attrition rate for NCEs entering clinical trials, is amatter of semantics. To do so, we need to change our definition ofwhat is considered ‘‘efficacious.’’ For example, currently most in

vivo MM models respond to therapy with drugs (NCEs, conven-tional therapies) administered as single agents. However, clin-icians would almost never treat their MM patients with only oneanti-MM agent because they are well aware that control of MM inthe clinical setting requires a combination of agents. Preclinically,we report that a drug effectively reduces MM growth when itreaches statistical significance of equal to or less than 5% (P < 0.05).Perhaps we need to make this more stringent as most single agentNCEs fail when tested in clinical trials. If we defined efficacy of aNCE (as a single agent) with more rigor, only the most effectivedrugs would enter clinical trials and would reduce the attritionrate. But are we simply concerned with lowering the attrition rateof NCEs entering clinical trials? It does not matter what theattrition rate is, what matters are the few effective drugs whichhave been discovered using the models and how patients havebenefited from them.

One cannot say that MM models are flawed because of theircurrent failure to predict clinical efficacy to the majority of NCEstested. There will never be one in vivo model of MM which willpredict the efficacy of most NCEs. Testing of NCEs has to beperformed on a variety of MM models before rushing into a clinicaltrial. Table 1 lists the various MM models currently available forthe testing of NCEs. Having stated this, we believe that MMxenografts will be the models which will more closely mimichuman MM in a clinically relevant manner. However, this will onlycome with the discovery of how to consistently grow primaryhuman MM tumors in immunocompromised mice with a hightransplantation success rate, and with many more additional yearsof basic research using these models. Only then can conclusions bemade as to the value MM xenograft models will have in predictingthe efficacy of NCEs and in their role in biomedical research.Opponents of MM models have raised the question as to why such‘‘flawed’’ models continue to dictate how biomedical research isconducted. What is flawed are not the in vivo MM models, but ourfailure to correctly translate the information we obtain from them,our definition of what an effective drug is and our concern with thelow predictive value of MM animal models (attrition rates) asopposed to focusing on the many patients who have benefited fromthe use of the already existing MM models.

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