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Ann Thorac Surg 1995;60:32-36
© 1995 The Society of Thoracic Surgeons

Successful Xenotransplantation of Human Lung Cancer Correlates With the Metastatic Phenotype

Jeremy R. Johnson, BA, William G. Hammond, MD, John R. Benfield, MD, Henry Tesluk, MD

Division of Cardiothoracic Surgery, University of California, Davis, School of Medicine, Sacramento, California


   Abstract
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 Footnotes
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background. Occult micrometastases could explain deaths from stage I non–small cell lung cancer (NSCLC) after complete resections. If patients who have occult metastases could be identified, systemic therapy might be beneficial.

Methods. Non–small cell lung cancers from 81 patients in stages I, II, and III were transplanted to nude beige mice. Mean follow-up was 22.5 months (2 to 61 months).

Results. Twenty-one xenotransplants successfully took, and seven metastasized in the nude mice. Neither the predominant cell type nor the incidence of lymph node metastases correlated with the results of xenotransplantation. Of the 21 patients whose NSCLCs took in xenotransplantation, 13 (61.9%) have had development of metastases, and 9 (42.9%) have died of the cancer. Among the 57 patients whose NSCLCs did not take, 14 (24.6%) have had development of metastases, and 9 (15.8%) have died of their cancer. The higher incidence of metastases in association with xenotransplant take is significant (p = 0.0032).

Conclusions. Patients whose NSCLCs take in xenotransplantation are at high risk for metastases. The xenotransplantation model is a step toward facilitating precise cellular biologic definition of the metastatic propensity of human NSCLC.


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 Introduction
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See also page 36.

Among patients with stage I (T1–2 N0 M0) non–small cell lung cancer (NSCLC), 20% to 30% eventually die of the systemic manifestations of metastases despite apparently complete resection of the primary neoplasms. This eventuality could be explained by the cancer cells' acquisition and expression of the metastatic phenotype [1] and the presence of occult micrometastases prior to pulmonary resection.

The current clinical management of NSCLC in stages I and II is resection followed by watchful waiting in the hope of cure. Systemic treatment with chemotherapy or combined treatment with irradiation and chemotherapy is reserved until there is evidence of recurrent cancer. There is now rapidly increasing evidence that modern induction therapy or neoadjuvant treatment can be effective in controlling the growth of NSCLC [2]. Thus, it is timely to seek methods to identify the presence of occult metastases in NSCLC patients with apparently curable cancers. Success in this endeavor would provide a basis for selecting a subset of patients with stage I resected cancers who should receive adjuvant systemic therapy.

With the exception of neoplasms like germ cell tumors for which there are well-established tumor markers, efforts to detect occult metastatic cancer have been frustrating. In vivo assays of the metastatic potential of primary cancers of the prostate, colon, ovary, and lung have been difficult and tedious [3–5]. Other attempts to accomplish the detection of occult metastases through in vitro studies of human bronchial cell lines could not predict the eventual appearance of overt metastases [6]. More recently, immunohistochemical studies of tumor-associated monoclonal antigens in resected NSCLC specimens correlated positively with patient survival times [7]. However, there is not yet a way to predict the eventual appearance of metastases.

We postulated that the metastatic phenotype of human NSCLC can develop during tumor propagation in nude mice. We further hypothesized that there is correlation between the propensity of NSCLC to metastasize in patients and the growth of NSCLC xenotransplants (XTs) in nude beige mice (NBM). Our purpose is to report a 5-year experience with xenotransplantation of human NSCLC. We shall show positive correlation between the growth of the xenografts and the occurrence of metastases.


   Material and Methods
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 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Harvesting of Original Cancer
Patients with NSCLC had pulmonary resections according to the usual criteria. The pathologists took all samples needed for diagnostic purposes. Residual specimens (according to a protocol originally approved in January 1989 and renewed in January 1994 by the institutional review board) were assessed for the presence and viability of NSCLC by frozen section or touch preparation. Viable specimens were placed in Dulbecco's phosphate-buffered saline solution without divalent cations at pH 7.4 (Gibco/Life Technologies) and transported on ice to our laboratory.

Procedure of Xenotransplantation
A representative sample of the cancer specimen to be transplanted was fixed in formalin to permit later histologic comparisons of the original transplant with the morphology of the successful XT. As previously described in detail [8], the solid tumor was coarsely minced in Dulbecco's phosphate-buffered saline solution containing 250 U/mL of penicillin, 0.5 mg/mL of streptomycin sulfate, and 0.05 mg/mL of gentamicin sulfate. The minced tumor was then aspirated through an 18-gauge needle and injected subcutaneously on the dorsum of the NBM hosts. If necessary, a wound clip was used to close the injection site. Xenotransplantation was ordinarily accomplished within 3 to 4 hours after operative devascularization of the primary cancer.

The XT Hosts
Male or female 6- to 8-week-old NBM (Charles River, Portage, ME; Harlan Sprague Dawley, San Diego, CA), which have congenitally reduced natural killer cell activity, were used as the XT recipients. The animals were housed 1 or 2 per cage on sterilized bedding in an AAALAC–approved facility and provided sterile water and sterile rodent chow ad libitum. All animals involved in this study received humane care in compliance with ``Guide for the Care and Use of Laboratory Animals'' published by the National Institutes of Health.

Evaluation of XT Growth
The mice were checked twice weekly after xenotransplantation for a minimum of 6 months for any palpable evidence of tumor growth. If no growth was noted after 6 months, the transplant was classified as a nontake. All palpable nodules were considered takes if they were confirmed histologically as cancer. The first take was considered as transplant generation (TG) 1 and a success in xenotransplantation.

Harvest of XTs and Propagation of Additional TGs
The procedures for propagation of tumor lines were previously described in detail [8] and are shown diagrammatically in Figure 1Go.



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  		Fig 1. . Tumor propagation. Serial transplant generations occurred when non–small cell lung cancer was transplanted from one nude beige mouse to another. Regrowth occurred after a successful take was incompletely excised or a portion of a tumor grew after reimplantation in the mouse from which it had been harvested. (Reprinted from Cancer Lett 1991;61:53–60 with permission of Elsevier Science Ireland Ltd.)

 
When xenografts reached 1 cm in greatest diameter, they were excised under aseptic conditions during general anesthesia induced with intraperitoneal administration of 240 mg/kg of tribromoethanol [9] or 80 mg/kg of sodium pentobarbital. At the time of harvest, an apparently viable sample was formalin fixed for histologic comparison with the primary neoplasm from which it arose and with subsequent XTs. The next TG in a new nude mouse host was initiated as previously described [10]. An ``established tumor line'' was any XT that grew beyond TG 1.

After each successful take was excised, a piece of tumor 1 to 2 mm3 was placed in the subcutaneous space of the mouse from which it had been harvested for the purpose of initiating regrowth. Successful regrowth was achieved when a newly palpable nodule was proved to be cancer by histologic evaluation.

A tumor growth cycle was defined as growth to 1 cm in diameter either from an initial inoculum of neoplasm in a new NBM or regrowth of a partially resected XT in the same host. Each tumor-bearing NBM was limited to five tumor growth cycles, after which time tumor hosts were killed humanely by exsanguination under general anesthesia.

A postmortem examination was conducted on each tumor-bearing mouse to detect macroscopic evidence suggestive of any metastatic lesions in the lung, lymphatics, liver, kidney, and gut. All metastases detected were evaluated histologically, as was the dorsal tumor from which they arose. Metastatic lesions that were found to exceed 5 mm in diameter were confirmed histologically and transplanted to new NBM hosts as already described.

Correlation Between Propagation and Clinical Outcome
The records of all patients involved in the study were reviewed. The histomorphology, degree of differentiation, and pathologic staging were noted for each patient. Clinical follow-up was derived from our own records or from primary care physicians. The presence or absence of metastases in each patient at postmortem examination or at the time of the most recent follow-up visit was determined.

Statistical Analysis
The significance of comparisons was determined using the Fisher exact t test with a 2 x 2 contingency table and the {chi}2 test [11].


   Results
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 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
We xenotransplanted portions of 81 freshly resected NSCLC from February 1989 to December 1994. The patients from whom the cancers were removed were 47 men and 34 women with a mean age of 63.9 years (range 36 to 81 years). At the time of writing, three XTs are less than 6 months old and therefore are too young to evaluate.

The 78 XTs came from 41 undifferentiated or poorly differentiated neoplasms and from 37 moderately or well-differentiated cancers. The predominant cell types and pathologic stages [12] are shown in Table 1Go.


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  		Table 1. . Pathologic Stage and Predominant Cell Type of 78 Human Non–Small Cell Lung Cancers Xenotransplanted to Nude Beige Mice
 
Results of Xenotransplantation
Of the 78 XTs, 21 took in TG 1, and 57 did not grow. Six XTs that grew in TG 1 did not progress further because the mice died before TG 2. Fifteen of the 21 successful XTs progressed to TG 3 to 8 and were considered established tumor lines.

Of these 15 established tumor lines, seven developed distant or regional metastases in the lung, lymphatics, liver, or kidneys of the nude mice. Among these seven lines, all metastases transplanted grew in serial transplantation. Figure 2Go shows a primary human NSCLC, the dorsal xenograft of the primary, and a lymph node metastasis that arose from that xenograft.



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  		Fig 2. . Morphology of non–small cell lung cancer during xenotransplantation and propagation in nude beige mice: (A) primary squamous cell carcinoma; (B) dorsal xenotransplant (transplant generation [TG] 6) derived from A; (C) lymph node metastasis in nude mouse that underwent postmortem examination when B was harvested; and (D) TG 1 growth of lymph node metastasis C in xenotransplantation. Note the stable morphologic features when the primary cancer is compared with TG 6 and with a distant metastasis that occurred after tumor propagation. (Hematoxylin and eosin; x100 before 22% reduction [A–C] or 38% reduction [D].)

 
The microscopic morphology of TG 1 cancers varied as greatly as the histology of the primary human NSCLCs from which they had come. Two of the 15 established tumor lines dedifferentiated to a spindle cell variant of squamous cell carcinoma. The remaining 13 demonstrated, at most, only one degree of change in differentiation. We confirmed the tendency for tumors to become less well differentiated during serial propagation as reported by other investigators [3] who used xenotransplantation models, but tumor morphology in our experiments remained remarkably consistent from one TG to another. Similarly, the metastatic lesions histologically examined closely resembled the dorsal XTs from which they arose.

Correlation of Propagation With Clinical Outcome
The 78 cancer patients from whom XTs were derived had a mean follow-up of 22.5 months (range, 2 to 61 months). Their metastatic lesions appeared in the usual locations such as brain, bones, and liver. The intervals between resection of the primary cancers and the appearance of metastases did not correlate with the results of xenotransplantation. Neither the predominant cell type of the original neoplasm (Table 2Go) nor the incidence of lymph node metastases at the time of pulmonary resection (Table 3Go) correlated with the results of xenotransplantation. To date, the establishment of tumor lines, regrowth of XTs in NBM, and metastases in the NBM have not correlated with clinical outcome.


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  		Table 2. . Correlation Between Predominant Cell Type of Human Non–Small Cell Lung Cancer and Success of Xenotransplantationa
 

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  		Table 3. . Relationship Between Lymph Node Metastases, Xenotransplantation Takes, and Development of Metastases in Patients After Resection of Non–Small Cell Lung Cancera
 
Table 4Go shows the correlation of XT takes with the appearance of metastases in the patients. Note that of the 21 patients whose NSCLCs took in xenotransplantation, 13 (61.9%), to date, have had development of overt metastatic lesions, and 9 (42.9%) of these patients have died of the cancer. Among the 57 patients whose NSCLCs did not take, 14 (24.6%), to date, have had metastases develop, and 9 (15.8%) of them have died as a result of the cancer. The higher incidence of metastases among patients whose cancers took in xenotransplantation compared with those whose cancers did not take is significant (p = 0.0032).


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  		Table 4. . Correlation Between Xenotransplantation Results and Metastasis in Patients With Non–Small Cell Lung Cancera
 

   Comment
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
We postulated that the metastatic phenotype can develop during tumor propagation in NBM. Our findings support this because metastases occurred in NBM before the humans in whom the cancers originated had any evidence of metastatic disease. It is not clear, however, whether the expression of the metastatic phenotype in nude mice, apparently independent of the behavior of the cancers in the patients, is a reflection of the development of this phenotype during xenotransplantation or an indication of its existence in the original NSCLCs prior to resection. Both possibilities exist. We believe it likely that the metastatic phenotype existed in unrecognized form in the NSCLCs in the patients. However, our experiments cannot yet address this issue definitively because the mean duration of follow-up is only 22.5 months, and we have no independent molecular or cellular markers of the metastatic phenotype.

Another hypothesis with which we began these experiments was that there is correlation between the propensity of NSCLC to metastasize in patients and the growth of derived XTs in nude mice. To support this hypothesis, one would expect that cancers that have already expressed the metastatic phenotype in patients through metastases to lymph nodes would be most likely to take in xenotransplantation. To date, new findings are contrary to expectations. However, the absence of positive correlation between the presence of lymph node metastases in patients and the take rate of NSCLCs in xenotransplantation does not negate the possible validity of our hypothesis because our short clinical follow-up and our still small experience with xenotransplantation preclude definitive assessment of this issue now. Additional experience may show that we have found a method to identify the metastatic phenotype while it is still occult to usual clinicopathologic methods of assessment.

Among the unexplained observations is the absence of correlation between the predominant NSCLC cell type and the success of xenotransplantation. We would have predicted that large cell undifferentiated carcinomas, which generally behave in a highly malignant fashion, would be more likely to take in transplantation than more differentiated cancers. We would also have expected positive correlation between the appearance of metastases in patients and the success of serial propagation in xenotransplantation. More experience of longer duration with experiments of the type we have described may reveal the expected correlations.

We have found compelling evidence of positive correlation between the propensity of NSCLC to metastasize in patients and the success of xenotransplantation. There has been a significantly higher incidence of metastases in patients whose cancers took in xenotransplantation compared with patients whose cancers did not take. We have essentially the same mean duration of follow-up in both groups of patients. Therefore, we believe this positive correlation to be meaningful and not a consequence of chance disparity between the two groups.

Our findings, although insufficient to justify therapeutic recommendations, suggest that patients whose cancers take in xenotransplantation are at high risk for metastases and that such patients may benefit from systemic therapy even in the absence of clinical evidence of metastases. These results are based on successful xenotransplantation 6 months after pulmonary resections, and we are the first to acknowledge that any assay that takes 6 months to complete is of limited clinical value. However, there are at least two ways in which our findings could eventually become useful in clinical practice. First, one could argue that 6 to 8 months of apparent cure after removal of NSCLC may still be time enough to begin effective systemic therapy for select patients who are at high risk for metastases. Second, and probably more important, we see this descriptive study of human NSCLC in xenotransplantation as a first step toward cellular biologic study of the metastatic propensity of NSCLC. We believe this model will be a valuable tool for study of the acquisition of the metastatic phenotype and that further use of the model will permit timely identification of this phenotype in NSCLCs that are apparently stage I.


   Footnotes
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 Footnotes
Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 
Presented at the Thirty-first Annual Meeting of The Society of Thoracic Surgeons, Palm Springs, CA, Jan 30–Feb 1, 1995.

Address reprint requests to Dr Benfield, Division of Cardiothoracic Surgery, University of California, Davis, Medical Center, 4301 X St, Sacramento, CA 95817.


   References
Top
 Footnotes
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

  1. Fidler IJ. Critical factors in the biology of human cancer metastasis: twenty-eighth G.H.A. Clowes memorial award lecture. Cancer Res 1990;50:6130–8.[Abstract/Free Full Text]
  2. Roth JA, Fossella F, Komaki R, et al. A randomized trial comparing perioperative chemotherapy and surgery with surgery alone in resectable stage IIIA non–small cell lung cancer. J Natl Cancer Inst 1994;86:673–80.[Abstract/Free Full Text]
  3. Houghton JA, Taylor DM. Maintenance of biological and biochemical characteristics of human colorectal tumours during serial passage in immune-deprived mice. Br J Cancer 1978;37:199–212.[Medline]
  4. Price JE, Zhang RD. Studies of human breast cancer metastasis using nude mice. Cancer Metastasis Rev 1990;8:285–97.[Medline]
  5. Lee C, Shervin DH, Kozlowski JM. In vivo and in vitro approaches to study metastases in human prostatic cancer. Cancer Metastasis Rev 1993;12:21–8.[Medline]
  6. Stevenson H, Gazdar AF, Phelps R, et al. Tumor cell lines established in vitro: an independent prognostic factor for survival in non–small-cell lung cancer. Ann Intern Med 1990;113:764–70.
  7. Miyake M, Taki T, Hitomi S, Hakomori S. Correlation of expression of H/Ley/Leb antigens with survival in patients with carcinoma of the lung. N Engl J Med 1992;327:14–8.[Abstract]
  8. Hammond WG, Benfield JR, Teplitz RL. Metastases from fresh human non–small cell lung cancers propagated in nude mice. Cancer Lett 1991;61:53–60.[Medline]
  9. Papaioannou VE, Fox JG. Efficacy of tribromoethanol anesthesia in mice. Lab Anim Sci 1993;43:189–92.[Medline]
  10. Hammond WG, Teplitz RL, Benfield JR. Lung cancer model for study of the metastatic process. Ann Thorac Surg 1991;52:732–7.[Abstract]
  11. Lee ET. Statistical methods for survival data analysis. Belmont, CA: Lifetime Learning Publications, 1980.
  12. Mountain CF. A new international staging system for lung cancer. Chest 1986;89:225S–33S.[Free Full Text]

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